4318

MMP9

matrix metallopeptidase 9;MMP9;matrix metallopeptidase 9

macrophage gelatinase|matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)|matrix metalloproteinase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)|matrix metalloproteinase-9|type V collagenase

20q13.12

protein-coding

OBJECTIVE: To construct differential expression profiles of adenoid cystic carcinoma cell lines for screening candidate genes related to metastasis and to verify some candidate genes in adenoid cystic carcinoma. METHODS: Restriction fragments differential display PCR (RFDD-PCR) was used to set up gene expression profiles of adenoid cystic carcinoma cell lines-ACC-M and ACC-2, with high and low metastasis potential respectively. Candidate genes were screened through bioinformatics analysis. Then, a gene family of these candidate genes was checked using semi-quantitative reverse transcription-PCR(RT-PCR). RESULTS: Two gene expression profiles including 5420 gene fragments were constructed, 12 genes of a family called matrix metalloproteinase genes (MMPs) were observed obvious differentially expressed between two cell lines. Results of semi-quantitative RT-PCR also identified this different expression of MMP2,MMP7,MMP9,MMP14,MMP15 and MMP24. CONCLUSION: The construction of gene expression profiles of ACC-M and ACC-2 cell lines makes the foundation for seeking the target genes of adenoid cystic carcinoma. MMP2,MMP7,MMP9 and MMP15 may be relevant with carcinogenesis, development and metastasis of adenoid cystic carcinoma, and different metastasis potential may result from different subtype of MMPs gene family.

BACKGROUND & OBJECTIVE: Previous researches of breast cancer mainly focused on its parenchyma and prognosis; however, the relationship between extracellular matrix of breast carcinoma and metastasis as well as prognosis was still unknown. The present study was designed to investigate the expression of matrix metalloproteinase 9 (MMP-9) and laminin- receptor (LM-R) in breast carcinoma and their correlation with tumor metastasis and prognosis. METHODS: The immunohistochemical method was applied to examine the expression of MMP-9 and LM-R in 80 samples of breast carcinoma and 10 breast tissues of hyperplasia. The absorbance(A) values that indicate the expression levels were analyzed by computer image technique. t-test, chi square-test and other related tests were used to analyze their relationship with tumor metastasis and prognosis. RESULTS: The expression of MMP-9 in breast carcinoma was apparently higher than that in hyperplasia (t=8.87, P< 0.05). LM-R expressed at the basal membrane and the edge of some acini and ducts in hyperplasia tissues, while expressed at cytoplasm and cellular membrane in breast carcinoma tissues. LM-R expression of tumor in 17 cases with LM-R expression in basal membrane was higher than that in those without LM-R expression in basal membrane (t=2.02, P< 0.05). LM-R expression was related to histological grade; the lower the degree of differentiation, the higher the expression level (F=3.27, P< 0.05). The expression of MMP-9 and LM-R related to lymphatic metastasis. The expression levels of MMP-9 and LM-R in the group which had more than 3 involved lymph node were lower than those in the group which had less than involved 3 lymph node and no involved lymph node (t(MMP-9)=3.42, t(LM-R)=4.31, P< 0.05). The expression of MMP-9 and LM-R in original focus had significantly positive relation to metastasis(r(MMP-9)=0.654, r(LM-R)=0.755, P< 0.001). MMP-9 had negative relation to progestogen receptor (PR) (r=-0.363,P< 0.05) and positive relation to LM-R (r=0.503,P< 0.01). The expression levels of MMP-9 and LM-R of the group which survived more than 3 years were lower than that in the group which survived less than 3 years (t(MMP-9)=3.57, t(LM-R)=4.22, P< 0.05). Both of MMP-9 and LM-R were negatively related to the survival of the patients with breast carcinoma (r(MMP-9)=-0.387, r(LM-R)=-0.369, P< 0.05). CONCLUSION: MMP-9 and LM-R are useful indexes for predicting the metastasis and prognosis of breast cancer.

expression of adhesion receptor integrin alphavbeta3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that alphavbeta3 cooperates with matrix metalloproteinase type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated by the activation state of the integrin. expression of activated alphavbeta3 in metastatic variants of MDA-MB 435 human breast cancer cells and primary metastatic cells from breast cancer patients strongly enhanced migration toward vitronectin and fibrinogen. This enhancement was mediated by a soluble factor produced by breast cancer cells expressing activated alphavbeta3. When transferred, this factor also up-regulated alphavbeta3-dependent migration of breast cancer cells that express the nonactivated integrin. The factor was identified as metalloproteinase MMP-9. Whereas all tested breast cancer cell variants produced latent MMP-9, only those with activated alphavbeta3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced alphavbeta3-dependent breast cancer cell migration. The migratory response was inhibited by tissue inhibitors of metalloproteinase or when MMP-9 was depleted from the inducing supernatants. The results indicate a causal relationship between the expression of activated integrin alphavbeta3 and production of enzymatically active MMP-9 in metastatic breast cancer cells. These molecules cooperate to enhance breast cancer cell migration toward specific matrix proteins, and this may contribute to the strongly enhanced metastatic capacity of breast cancer cells that express activated alphavbeta3.

In order to study the expression of MMP2, MMP3 and MMP9 in breast cancer brain metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain. Immunohistochemistry and Western Blotting analyses showed that MMP-2, -3 and -9 proteins expressions are consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. These results were confirmed by RT-PCR. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. Together our results suggest that MMP-2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain.

OBJECTIVE: To investigate effects of P-glycoprotein (gp) substrate drugs on the expression of CD147 and MMP2 and 9 in multidrug resistant breast cancer cells. METHODS: MDR human breast cancer cell line, MCF7/AdrR, and its sensitive parental line, MCF7, were treated with various concentrations of P-gp substrate drugs, including paclitoxel and vincristine, and P-gp nonsubstrate drugs, bleomycin, in serum-free media. At the end of the treatment, expressions of CD147 and MMP2 and 9 were determined by real-time PCR and western blot. RESULTS: Increased expressions of CD147 and MMP2 and 9 were observed in multidrug resistant cancer cells compared with their parental MCF7 cells. After treatment with bleomycin, the expression of CD147 and MMP2 and 9 in both MCF7 and MCF7/AdrR cells remained unchanged (P > 0.05). However, treatment with paclitoxel and vincristine resulted in a remarkable over-expression of CD147 and MMP2 and 9 at both transcription and protein levels in MCF7/AdrR cell line (P < 0.05), while MCF7 cells failed to show similar response. CONCLUSIONS: P-gp substrate drugs can greatly upregulate the expression of CD147 and MMP2 and 9 in multidrug resistant breast cancer cells, therefore enhancing the tumor metastatic capability.

Surgery is still the main curative therapeutic modality for breast cancer. Although surgery often results in the successful removal of the primary tumor, its process could increase the risk of metastases of residual cancer cells. Understanding of the connection between breast cancer metastasis and surgical wound will lead to the establishment of a proper treatment strategy for postoperative cancer patient. AIM: To study the influence of surgical procedure on the metastasis of primary breast cancer. METHODS: We established MDA-MB-435 human breast cancer xenograft model. Levels of Pro-matrix metalloproteinase 9 (Pro-MMP-9) and vascular endothelial growth factor (VEGF) in host serum and tumors were tested at different time points with ELISA and zymography and correlated to tumor growth and postoperative metastasis. RESULTS: Our study demonstrated surgical wound had promoting effect on tumor growth and pulmonary metastasis of human breast cells, if tumor cells remain in bodies. This effect might be related to the postoperative interaction of cancer and host cells, which resulted in expression of Pro-MMP-9. Surgical process could also increase the VEGF expression in tumor tissues. CONCLUSIONS: Surgical wound-produced host Pro-MMP-9 and tumor cell VEGF might be important mediators leading to metastasis of residual breast cancer after surgery.

OBJECTIVE: To study the expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF)-C in breast cancer and their role in lymph node metastasis. METHODS: Immunohistochemical staining was used to detect the expression of VEGF-C, MMP-2, MMP-9 and LYVE-1 in 84 cases of breast cancer, including 52 cases with and 32 cases without lymph node metastases. The recombinant vector (pSIREN-VEGF-C) was transfected into human breast cancer cell MCF-7 by liposome, and the RNA expression of MMP-2, MMP-9 and VEGF-C in MCF-7 cells after transfection was detected by PCR. RESULTS: The expressions of MMP-2, MMP-9 and VEGF-C were 98.1% (51/52), 88.5% (46/52), and 94.2% (49/52) respectively for the metastatic group, and 75.0% (24/32), 53.1% (17/32), and 65.6% (21/32) respectively for the non metastatic group, and there was significant difference between these groups (P < 0.05). The lymphatic vessel density between these two groups was also significantly different (P < 0.05). Increased expression of MMP-2, MMP-9 and VEGF-C was also associated with increased number of lymphatic vessels had also increased (P < 0.05). The expression of MMP-2, MMP-9 and VEGF-C in MCF-7 cells after gene transfection decreased significantly (P < 0.05). CONCLUSION: MMP-2 and MMP-9 in conjunction with VEGF-C, promote lymphangiogenesis and lymph node metastasis of breast cancer.

PURPOSE: BMP-6, which belongs to the TGF-beta superfamily, is a multifunctional molecule with distinct abilities in embryogenesis and organogenesis. Our recent research has implied that BMP-6 may suppress breast cancer metastasis. In the present study, we extended to elucidate the molecular mechanism by which BMP-6 exerts its anti-tumorigenic effect. METHODS: The Boyden chamber assay was used to examine the ability of BMP-6 and HO-1 in MCF-7 malignant progress. RT-PCR, western blot, luciferase assay, and quantitative CHIP were used to determine the potential mechanism and signaling pathways by which BMP-6 and HO-1 function as anti-metastatic factors in MCF-7 cells. RESULTS: The Boyden chamber assay showed that BMP-6 inhibited the migration and invasion of MCF-7 cells, which effect was significantly deprived by knockdown of HO-1. We further demonstrated that BMP-6 treatment resulted in an activation of HO-1 transcription through the recruitment of Smad1/5 to the Smad-responsive element on its promoter. In addition, BMP-6-induced up-regulation of HO-1 exhibited an inhibitory effect on MMP-9 secretion in a paracrine action in MCF-7 cells. Overexpression of BMP-6 and HO-1 synergistically suppressed MMP-9 transcription, which effect was specifically mediated via the MAPK/p38/AP-1 signaling. However, blockade of HO-1 using ZnPPIX totally abolished BMP-6-regulated MMP-9 activation in MCF-7 cells. CONCLUSIONS: These observations suggest a novel role of BMP-6/HO-1 cascade to relieve breast cancer metastasis by regulating the secretion of growth factors in tumor microenvironment.

Epithelial to mesenchymal transition (EMT) and extracellular matrix degradation are critical for the initiation and progression of tumor invasion. We have recently identified Kruppel-like factor 8 (KLF8) as a critical inducer of EMT and invasion. KLF8 induces EMT primarily by repressing E-cadherin transcription. However, how KLF8 promotes invasion is unknown. Here, we report a novel KLF8-to- matrix metalloproteinase (MMP)9 signaling that promotes human breast cancer invasion. To identify the potential KLF8 regulation of MMPs in breast cancer, we established two inducible cell lines that allow either KLF8 overexpression in MCF-10A or knockdown in MDA-MB-231 cells. KLF8 overexpression induced a strong increase in MMP9 expression and activity as determined by quantitative real-time PCR and zymography. This induction was well correlated with the MMP inhibitor-sensitive Matrigel invasion. Conversely, KLF8 knockdown caused the opposite changes that could be partially prevented by MMP9 overexpression. Promoter-reporter assays and chromatin and oligonucleotide precipitations determined that KLF8 directly bound and activated the human MMP9 gene promoter. Three-dimensional (3D) glandular culture showed that KLF8 expression disrupted the normal acinus formation, which could be prevented by the MMP inhibitor, whereas KLF8 knockdown corrected the abnormal 3D architecture, which could be protected by MMP9 overexpression. KLF8 knockdown promoted MDA-MB-231 cell aggregation in suspension culture, which could be prevented by MMP9 overexpression. KLF8 knockdown inhibited the lung metastasis of MDA-MB-231 cells in nude mice. Immunohistochemical staining strongly correlated the co-expression of KLF8 and MMP9 with the patient tumor invasion, metastasis and poor survival. Taken together, this work identified the KLF8 activation of MMP9 as a novel and critical signaling mechanism underlying human breast cancer invasion and metastasis.

BACKGROUND: The c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells. RESULTS: Ectopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP) 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed the growth of mammary tumors in BALB/c mice and affected the metastatic potential of breast cancer cells in an organ-specific manner. CONCLUSIONS: This study identified c-Myb as a matrix-dependent regulator of invasive behavior of breast cancer cells.

OBJECTIVE: To investigate possible mechanism of silencing lysyl oxidase (LOX) gene by RNA interference affecting on invasion and metastasis of breast cancer cells. METHODS: LOX-RNAi-LV was designed and synthesized, which was transfected into breast cancer cell line MDA-MB-231. The expressions of LOX, MMP-2 and MMP-9 were determined by Real-time PCR in MDA-MB-231 cells, and the protein expression of LOX was determined by Western blot. The cells migration and invasion abilities were measured by cell migration and invasion test. 111 cases of breast cancer tissue and cancer-adjacent breast tissues and 20 cases of benign lesion tissues of LOX, MMP-2 and MMP-9 were detected by immunohistochemistry, and the relationship of LOX and clinicopathological characteristics was analyzed. RESULTS: The expression levels of LOX mRNA and protein were down-regulated obviously after transfecting LOX-RNAi-LV, with the inhibition rate 89.2% +/- 1.3% and 84.4% +/- 0.4% respectively. The relative expressions of MMP-2 and MMP-9 mRNA were 0.496 +/- 0.021 and 0.571 +/- 0.099 in RNAi group, which was significantly lower than that in negative control group (0.846 +/- 0.047, 0.786 +/- 0.042) and blank control group (1.000 +/- 0.000, 1.000 +/- 0.000) (both P < 0.05). Cell migration and invasion test showed the average cell numbers per field in the group RNAi were 47 +/- 2 and 63 +/- 2, was significantly lower than that in negative control group (100 +/- 1, 118 +/- 2) and blank control group (100 +/- 1, 118 +/- 2) (both P < 0.05). The expression of LOX protein in breast cancer, cancer-adjacent breast tissues and benign breast tumor were 48.6% (54/111), 26.1% (29/111), 20.0% (4/20), the expression of LOX protein in breast cancer was significantly higher than that in cancer-adjacent breast tissues and benign lesion tissues (P = 0.019). The expression of LOX protein was associated with clinical stage, lymph node metastasis, tumor size. Correlation analysis showed that LOX protein expression was significantly positive correlation with MMP-2 (r = 0.262, P = 0.005) and MMP-9 (r = 0.424, P = 0.000). CONCLUSION: LOX can promote invasion and metastasis of breast cancer; LOX and MMP-2, MMP-9 may have a synergistic role in promoting invasion and metastasis of breast cancer.

The aim of this study was to investigate expression of CD147 and MMP-9 in triple-negative breast cancer (TNBC) so as to determine whether these two proteins may be correlated with poor prognosis of TNBC patients. We examined the expression levels of the CD147 and MMP-9 in 127 patients with TNBC and 30 patients with mammary gland fibroma using immunohistochemical staining before any treatments. Furthermore, we analyzed the correlation between the expression of these two proteins and various clinicopathologic factors including survival status of patients with TNBC. Positive stain of CD147 and MMP-9 was observed in all samples of TNBC. A statistically positive correlation was observed between the expression levels of CD147 and MMP-9 in TNBC tissues. The incidences of high expression were 48.0 % for CD147 and 53.5 % for MMP-9 in 127 TNBC tissues, respectively. High expression of either CD147 or MMP-9 was significantly correlated with clinical feature and shorter progression-free survival (PFS) (P(CD147) = 0.039; P(MMP-9) = 0.017) and overall survival (OS) (P(CD147) = 0.037; P(MMP-9) = 0.023). The expression levels of CD147 and MMP-9 are positively correlated with invasion, metastasis and shorter PFS/OS of TNBC. Patients with high expression of CD147 and MMP-9 had poor prognosis than TNBC patients with low expression.

Breast cancer is the leading cause of cancer deaths among women worldwide, including Thailand. In the present study, the differential mRNA expression of SVEP1, LPHN3, KLB, ITGA7, SEMA3G, TNS1 and MMP13 genes was examined in breast cancer using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). Among these genes, increased LPHN3 and MMP13 mRNA expression levels correlated with axillary-node metastasis (P=0.02). Multiple logistic regression analysis revealed that LPHN3 and MMP13 mRNA expression is significantly associated with axillary node status in breast cancer (P=0.04).

Invadopodia-dependent degradation of the basement membrane plays a major role during metastasis of breast cancer cells. Basement membrane degradation is mediated by targeted secretion of various matrix metalloproteinases (MMPs). Specifically, MMP2 and MMP9 (MMP2/9) possess the ability to hydrolyze components of the basement membrane and regulate various aspects of tumor growth and metastasis. However, the membrane transport machinery that mediates targeting of MMP2/9 to the invadopodia during cancer cell invasion remains to be defined. Because Rab GTPases are key regulators of membrane transport, we screened a human Rab siRNA library and identified Rab40b GTPase as a protein required for secretion of MMP2/9. We also have shown that Rab40b functions during at least two distinct steps of MMP2/9 transport. Here, we demonstrate that Rab40b is required for MMP2/9 sorting into VAMP4-containing secretory vesicles. We also show that Rab40b regulates transport of MMP2/9 secretory vesicles during invadopodia formation and is required for invadopodia-dependent extracellular matrix degradation. Finally, we demonstrate that Rab40b is also required for breast cancer cell invasion in vitro. On the basis of these findings, we propose that Rab40b mediates trafficking of MMP2/9 during invadopodia formation and metastasis of breast cancer cells.

Matrix metalloproteinases (MMPs) have been implicated in diverse roles in breast cancer development and progression. While many of the different MMPs expressed in breast cancer are produced by stromal cells MMP-9 is produced mainly by the tumor cells themselves. To date, the functional role of tumor cell-produced MMP-9 has remained unclear. Here, we show that human breast cancer cell-produced MMP-9 is specifically required for invasion in cell culture and for pulmonary metastasis in a mouse orthotopic model of basal-like breast cancer. We also find that tumor cell-produced MMP-9 promotes tumor vascularization with only modest impact on primary tumor growth, and that silencing of MMP-9 expression in tumor cells leads to an altered transcriptional program consistent with reversion to a less malignant phenotype. MMP-9 is most highly expressed in human basal-like and triple negative tumors, where our data suggest that it contributes to metastatic progression. Our results suggest that MMP9 may offer a target for anti-metastatic therapies for basal-like triple negative breast cancers, a poor prognosis subtype with few available molecularly targeted therapeutic options.

BACKGROUND & OBJECTIVE: Studies of tumor invasion and metastasis have focused on the degradation of extracellular matrix (ECM). Matrix metalloproteinases (MMPs), which could degrade ECM, were implicated in cancer invasion and metastasis. Activated MMPs were controlled by specific tissue inhibitors of metalloproteinases (TIMPs). This study was designed to investigate the expression of MMP-2, MMP-9 and TIMP-1, TIMP-2 in human squamous cell carcinomas of uterine cervix, and the association with invasion and metastasis of human squamous cell carcinoma of uterine cervix. METHODS: Tissue samples from 40 cases of squamous cell carcinoma of the uterine cervix, 29 cases of cervical intraepithelial neoplasia (CIN), and 16 cases of normal cervices were stained immunohistochemically. RESULTS: Immunohistochemical staining of tumor cells for MMP-2, MMP-9, TIMP-1, and TIMP-2 were noted 82.5%, 70.0%, 62.5%, and 97.5% in carcinomas, 44.8%, 62.1%, 86.21%, and 96.55% in CIN lesions respectively, for MMP-2, MMP-9, TIMP-1 in 25.0% normal cervices, but for TIMP-2 in 56.25% normal cervices. There was significant difference for MMP-2 among carcinomas, normal cervices, and CIN respectively (P < 0.01). There was significant difference for MMP-9 among normal cervices, carcinomas, and CIN, respectively (P < 0.05). The positive rates of TIMP-1 and TIMP-2 in the cervical carcinoma group and CIN group were obviously higher than that in normal cervical group(P < 0.01). The positive rates of MMP-2 and MMP-9 in lymph node metastasis were obviously higher than that in without lymph node metastasis(P < 0.01), but for TIMP-2 was contrary and there was no significant difference for TIMP-1. No significant correlation could be established among the expression of these markers and the tumor diameter, the clinical stage, and pathologic grade. CONCLUSIONS: The over-expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 may play a key role in invasion and lymph-node metastasis of in squamous carcinoma of the cervix.

OBJECTIVE: To investigate the expressions of COX-2 and MMP-9 in cervical carcinoma and their clinical significance. METHODS: Immunohistochemical SP method was used to detect the expressions of COX-2 and MMP-9 in 72 cases of invasive carcinoma of cervix (ICC) and 16 cases of normal cervical epithelium remote from tumor (NCE), and the relationship between the expressions of COX-2 and MMP-9 in ICC and some factors relating to clinical pathology of cervical carcinoma such as histopathological grading, lymph node metastasis, stroma involvement and FIGO staging were analyzed statistically. RESULTS: The rates of positive expression of COX-2 and MMP-9 in ICC were significantly higher than those in NCE. COX-2: 88.9% in group ICC and 12.5% in group NCE, P = 0.000. MMP-9: 94.4% in group ICC and 43.8% in group NCE, P = 0.000. The expression of COX-2 was positively correlated with lymph node metastasis and stroma involvement (r = 0.296, P = 0.012 in group ICC and r = 0.257, P = 0.029 in group NCE, respectively). The expression of MMP-9 was positively correlated with FIGO staging (r = 0.329, P = 0.005) and histopathological grading (r = 0.351, P = 0.003). The expression of COX-2 was positively correlated with the expression of MMP-9 in ICC (r = 0.297, P = 0.011). CONCLUSION: The overexpressions of COX-2 and MMP-9 are closely related to the invasion and growth of cervical carcinoma. The tissue with overexpression of COX-2 has strong invasion ability. COX-2 and MMP-9 have synergistic effect on proliferation, invasion and metastasis of cancer cells. Detecting the expression of both COX-2 and MMP-9 may be of value in further understanding the biological behavior and predicting the prognosis of cervical carcinoma.

The BRCA1-interacted transcriptional repressor ZBRK1 has been associated with antiangiogenesis, but direct evidence of a tumor suppressor role has been lacking. In this study, we provide evidence of such a role in cervical carcinoma. ZBRK1 levels in cervical tumor cells were significantly lower than in normal cervical epithelial cells. In HeLa cervical cancer cells, enforced expression inhibited malignant growth, invasion, and metastasis in a variety of in vitro and in vivo assays. expression of the metalloproteinase MMP9, which is known to be an important driver of invasion and metastasis, was found to be inversely correlated with ZBRK1 in tumor tissues and a target for repression in tumor cells. Our findings suggest that ZBRK1 acts to inhibit metastasis of cervical carcinoma, perhaps by modulating MMP9 expression.

Extracellular proteolysis mediates tissue homeostasis. In cancer, altered proteolysis leads to abnormal tumor growth, inflammation, tissue invasion, and metastasis. Matrix metalloproteinase-9 (MMP-9) represents one of the most prominent proteinases associated with inflammation and tumorigenesis. The recently identified human transcription factor sLZIP is a member of the leucine zipper transcription factor family. Although sLZIP is known to function in ligand-induced transactivation of the glucocorticoid receptor, its exact functions and target genes are not known. In this study, we investigated the role of sLZIP in MMP-9 expression and its involvement in cervical cancer development. Our results show that sLZIP increased the expression of MMP-9 at both the mRNA and protein levels and the proteolytic activity of MMP-9 in HeLa and SiHa cells. sLZIP also increased the transcriptional activity of MMP-9 by binding directly to the cAMP-responsive element of the MMP-9 promoter region. Involvement of sLZIP in MMP-9 expression was further supported by the fact that ME-180 cells expressing sLZIP siRNA were refractory to MMP-9 expression. Results from wound healing and invasion assays showed that sLZIP enhanced both the migration and invasion of cervical cancer cells. The increased migration and invasion of HeLa and SiHa cells that were induced by sLZIP were abrogated by inhibition of the proteolytic activity of MMP-9. These results indicate that sLZIP plays a critical role in MMP-9 expression and is probably involved in invasion and metastasis of cervical cancer.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of cancers. However, the effect of CCL2 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CCL2 increased the migration and expression of matrix metalloproteinase (MMP)-9 in human chondrosarcoma cells. CCL2-mediated migration and MMP-9 up-regulation were attenuated by CCR2, Ras, Raf-1, and MEK inhibitor. Activation of the Ras, Raf-1, MEK, ERK, and NF-kappaB signaling pathway after CCL2 treatment was demonstrated, and CCL2-induced expression of MMP-9 and migration activity were inhibited by the specific inhibitor, and mutant of Ras, Raf-1, MEK, ERK, and NF-kappaB cascades. Taken together, our results indicated that CCL2 enhances the migration of chondrosarcoma cells by increasing MMP-9 expression through the CCR2 receptor, Ras, Raf-1, MEK, ERK, and NF-kappaB signal transduction pathway.

AIM: To explore the role of the matrix metalloproteinase-9 (MMP-9) polymorphism in colorectal cancer (CRC) in a northeast Chinese population. METHODS: Genotyping of MMP-9-1562C>T and 279R>Q polymorphisms was carried out on blood samples from 137 colorectal cancer patients and 199 controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Multivariate logistic regression models were used to calculate adjusted odds ratios (OR) and 95% confidence intervals (95% CI). RESULTS: The distribution of MMP-9 -1562C>T and 279 R>Q genotype was not significantly associated with the risk of CRC. However, the risk of llymph node metastasis of CRC was increased in patients with the -1562T allele (OR = 2.601; 95% CI = 1.160-5.835; P = 0.022). The frequency of MMP-9 279RR + RQ genotype was higher than the QQ genotype among CRC patients younger than sixty years old (OR = 0.102; 95% CI = 0.013-0.812; P = 0.012). CONCLUSION: Our results indicated that the MMP-9-1562C>T polymorphism affects lymph node metastasis of CRC. In addition, the MMP-9 279R allele may lead to a younger age of onset of colorectal cancer.

Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and membrane-type matrix metalloproteinase 1 (MT1-MMP) are involved in colorectal cancer invasion and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) inhibits MMP-2, MMP-9 and MT1-MMP. We examined the clinicopathological significance of the relative expression of these genes in patients with colorectal cancer, especially with regard to metastasis. We studied surgical specimens of cancer tissue and adjacent normal mucosa obtained from 205 patients with untreated colorectal carcinoma. MMP-2, MMP-9, MT1-MMP, RECK and beta-actin mRNA of cancer tissue and adjacent normal mucosa were measured by quantitative real-time reverse-transcriptase polymerase chain reaction. MT1-MMP gene expression was higher in cancer tissue than in adjacent normal mucosa. In contrast, MMP-2, MMP-9 and RECK gene expression levels were lower in cancer tissue than in adjacent normal mucosa. As for the relationship between the gene expression and clinicopathological factors, MMP-2 expression correlated with the depth of invasion, venous invasion and liver metastasis; MMP-9 and RECK expression correlated with venous invasion. There were positive correlations among the gene expression levels of MMP-2, MMP-9 and RECK. MMP-2 gene expression was considered a useful predictor of liver metastasis from colorectal cancer.

The role of expression of markers (beta-catenin, matrix metalloproteinase 9, collagen IV, and laminin) in primary colorectal adenocarcinomas and their metastases in the liver and lymph nodes of patients with colorectal cancer was studied. High level of matrix metalloproteinase 9 expression in zones of invasive growth of colorectal cancer was associated with high accumulation of beta-catenin in cancer cell nuclei in the peripheral zones of 30% studied tumors. The presence of nuclear beta-catenin and high content of matrix metalloproteinase 9 in the tumor were associated with abnormal accumulation of laminin in the cytoplasm and with the absence of basal membranes containing collagen IV. These changes were characteristic of colorectal cancer with high invasive metastatic potential. It was found that beta-catenin, matrix metalloproteinase 9, laminin, and collagen IV were important markers for prediction of the clinical course of colorectal cancer. The expression of proteins associated with risk of metastases in the liver was coordinated and most pronounced in zone of invasive front-line of tumors.

Colorectal cancer mortality largely reflects metastasis, the spread of the disease to distant organs. Matrix metalloproteinase 9 (MMP-9) is a key regulator of metastasis and a target for anticancer strategies in colon cancer. Here, the overexpression of MMP-9 in pure tumor epithelial, but nor stromal, cell populations was associated with metastatic progression of colorectal cancer, as defined by reverse transcriptase-polymerase chain reaction (qRT-PCR) and confirmed by immunostaining. Thus, cancer cell MMP-9 represents a novel, selective prognostic and predictive factor that may be exploited for more effective disease stage stratification and therapeutic regimen selection in patients with colorectal cancer.

CONTEXT: Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) preferentially degrade the basement membrane, a key step in tumor invasion, metastases, and induction of vascularization of tumor tissue. AIM: To determine MMP-2 and MMP-9 in situ mRNA expressions in colorectal adenocarcinomas from Iraqi patients. MATERIALS AND METHODS: Forty archived paraffin-embedded colorectal adenocarcinoma samples and their resection margins were enrolled in our study. Thin paraffin embedded sections (3-5 mum thick) of both tumor and resection margins were prepared for each respective biopsy and were used to detect MMP-2 and MMP-9 in situ mRNA expressions based on in situ hybridization technique. STATISTICAL ANALYSIS: Statistical analyses were conducted to describe different variables and parameters in this research, and to describe relationships with each other as well. For the comparisons, the t test of significance was used. The associations were found by chi-square (chi2 ) and analysis of variance (ANOVA) tests, or as appropriate, as well as 95% confidence interval. The correlations were calculated using correlation coefficient (r). Statistical significance was defined as P < 0.05. RESULTS: Based on the current outcome, there were significant differences in MMP-2 and MMP-9 in situ mRNA expressions when each tumor sample were compared to its respective resection margin (P < 0.001 and P < 0.001, respectively). When tumor samples were analyzed based on their depth of invasion, means of in situ mRNA expressions of both MMP-2 and MMP-9 were significantly increased in the group in which tumor invaded submucosa into muscularis properia (SMP) compared to that of tumor in serosa (SE) group and tumor invading other organs (OR) group (P < 0.05 and P < 0.05, respectively). Furthermore, when tumor lymph node metastases were questioned, exclusively, MMP-2 in situ mRNA expression was significantly differentiated among N0, N1, and N2 groups (P < 0.05). Regarding the possible correlation between the two investigated parameters with respect to various histopathological variables, both MMP-2 and MMP-9 in situ mRNA expressions have significant positive correlation in tumor samples (r = 0.88), whereas in resection margins, this correlation was absent. Interestingly, MMP-2 and MMP-9 in situ mRNA expressions were found to correlate positively as well as significantly within tumor differentiation [well-differentiated (WD) adenocarcinoma: r = 0.78; moderately differentiated (MD) adenocarcinoma: r = 0.90; and poorly differentiated (PD) adenocarcinoma: r = 0.91], tumor stage (A-B: r = 0.70 and C-D: r = 0.95), depth of invasion (SMP: r = 0.77; SE: r = 0.87; and OR: r = 0.97), lymph node metastasis (N0: r = 0.82; N1: r = 0.92; and N2: r = 0.96), and tumor size (<3 mm 3 : r = 0.76 versus >/=3 mm3: r = 0.94). CONCLUSIONS: Overexpression of MMP-2 and MMP-9 are often associated with increased invasive metastatic potential of colorectal adenocarcinoma. However, their activities are essential during the early stages of tumor progression.

BACKGROUND: The wnt/beta-catenin signaling pathway is known to affect in cancer oncogenesis and progression by interacting with the tumor microenvironment. However, the roles of wnt3a and wnt5a in colorectal cancer (CRC) have not been thoroughly studied. In the present study, we investigated the expression of wnt protein and the concordance rate in primary tumor and metastatic sites in CRC. To determine the relationship of wnt proteins with invasion related protein, we also analyzed the association between wnt protein expression and the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor receptor-2 (VEGFR-2). METHODS: tumor tissue was obtained from eighty-three paraffin- embedded blocks which were using resected tissue from both the primary tumor and metastatic sites for each patient. We performed immunohistochemical staining for wnt3a, wnt5a, beta-catenin, MMP-9 and VEGFR-2. RESULTS: Wnt3a, wnt5a, beta-catenin, and MMP-9 expression was high; the proteins were found in over 50% of the primary tumors, but the prevalence was lower in tissue from metastatic sites. The concordance rates between the primary tumor and metastatic site were 76.2% for wnt5a and 79.4% for wnt3a and beta-catenin, but VEGFR-2 was expressed in 67.4% of the metastatic sites even when not found in the primary tumor. Wnt3a expression in primary tumors was significantly associated with lymph node involvement (p = 0.038) and MMP-9 expression in the primary tumor (p = 0.0387), mesenchyme adjacent to tumor (p = 0.022) and metastatic site (p = 0.004). There was no other relationship in the expression of these proteins. Vascular invasion in primary tumor tissue may be a potential prognostic marker for liver metastasis, but no significant association was observed among the wnt protein, MMP-9, and VEGFR-2 for peritoneal seeding. In survival analysis, beta-catenin expression was significantly correlated with overall survival (p = 0.05). CONCLUSIONS: Wnt3a and wnt5a expression had a concordance rate higher than 60% with a high concordance rate between the primary tumor and metastatic site. Wnt3a expression is associated with the expression of MMP-9 in primary tumor tissue adjacent mesenchymal tissue, and at the metastatic site. As a prognostic marker, only beta-catenin expression showed significant relation with survival outcome.

The molecular mechanism underlying the invasiveness of colorectal cancer (CRC) cells remains largely unknown. Here, we found that astrocyte elevated gene-1 (AEG-1) was significantly upregulated in CRC tissues, compared with the adjacent normal tissues from human patients. Ectopic expression of AEG-1 enhanced the invasive ability of CRC cells, while small interfering RNA (siRNA)-induced knockdown of AEG-1 inhibited the invasive ability of CRC cells. Transcription, protein levels, and secretion of matrix metalloproteinase 9 (MMP9), all increased by AEG-1 overexpression in CRC cells, and all decreased by AEG-1 inhibition. Suppression of endogenous MMP9 abrogated the effects of AEG-1 on invasiveness, without affecting AEG-1 levels. Taken together, these findings suggest that AEG-1 contributes to CRC invasiveness and metastasis by enhancing MMP9 activity. Thus, AEG-1 appears to be a novel therapeutic target for preventing the metastasis of CRC.

Matrix metalloproteinase 9 (MMP9) and CD147 play a role in invasion and metastasis of many types of human malignancies. The correlation of the expression of MMP9 and CD147 with invasion and metastasis of invasive squamous cell carcinoma (SCC) of the uterine cervix has not been examined. In the present study, RT-PCR assay was used to detect the expression level of MMP9 mRNA semiquantitatively, and immunohistochemical stain was adapted to evaluate the score of CD147 on the cell membrane or in the cytoplasm of tumor cells of 65 cases of invasive squamous cell carcinoma of the uterine cervix and 21 cases of chronic cervitis tissues. MMP9 and CD147 expression in correlation with invasion, metastasis, and differentiation of invasive SCC of the uterine cervix was analyzed statistically. We found that MMP9 and CD147 expression was elevated significantly in tumor tissue compared to the control (cervical epithelium of chronic cervitis) (P<0.01). In the comparison of MMP9 and CD147 expression in 47 cases with lymph node metastasis and 18 cases without lymph node metastasis, there was a significantly higher expression of MMP9 and CD147 in the group with lymph node metastasis (P<0.05 for MMP9, P<0.01 for CD147). MMP9 expression was significantly higher in 24 cases of poor differentiation than in 41 cases of moderate differentiation (P<0.05). No difference was found in CD147 expression between poor and moderate differentiation (P>0.05). No significant difference in MMP9 and CD147 expression levels was obtained between 26 cases of FIGO stage I tumors and 39 cases of stage II tumors (P>0.05 for MMP9, P>0.05 CD147). There was no correlation between MMP9 or CD147 expression levels and the resected tumor size (P>0.05). The positive correlation (r=0.568, P<0.001) of MMP9 expression and CD147 score was seen in the tumor tissues of 65 cases. The data in this study show that MMP9 and CD147 expression are correlated with invasion, metastasis of squamous cell carcinoma of the uterine cervix, and that MMP9 expression is correlated with poor differentiation of invasive squamous cell carcinoma of the uterine cervix.

BACKGROUND & OBJECTIVE: Matrix metalloproteinase-9 (MMPs) and its tissue inhibitor of metalloproteinase-1 (TIMPs) have the important regulatory roles in the processes during tumor invasion and metastasis. Observing the coexpression of matrix metalloproteinase 9(MMP-9) and tissue inhibitor of metalloproteinase 1(TIMP-1) in gastric carcinomas, this study was designed to investigate the relationship of the expression unbalance of MMP-9 and TIMP-1 to the invasiveness, metastasis, and prognosis in gastric carcinomas. METHODS: By using immunohistochemistry, the expression of MMP-9, TIMP-1, and proliferating cell nuclear antigen Ki-67 was detected in 256 gastric cancers, and the patients were followed-up. RESULTS: The expressive degree of MMP-9 in the tumor invasion beyond musculariis propria(70.13%) was higher than that within lamina propria or submucosa(42.50%, P < 0.05) and its expression in tumor was positively associated with tumor cell proliferation index and lymph node metastasis, and its expression in high TNM stage(75.00%) was higher than those in low TNM stage(46.15%, P < 0.05). The relation of the expression of TIMP-1 to the depth of invasion, lymph node metastasis, and TNM stage was negative(Pearson contingency coefficient were 0.1688, 0.3556, and 0.3004, respectively P < 0.05). There were four groups of the unbalance expression of MMP-9 and TIMP-1 in gastric carcinomas. While the frequency of the serosa invasion and lymph node metastasis in the group of the expression of MMP-9 over TIMP-1 was significantly higher than those in the groups of the expression of TIMP-1 over MMP-9 or the balance expression of MMP-9 and TIMP-1. However, prognosis of the patients with the expression of MMP-9 over TIMP-1 was significantly worse than that with expression of TIMP-1 over MMP-9(P < 0.05). CONCLUSION: The expression of MMP-9 was positively related to the tumor invasiveness and metastasis in gastric cancers, however the association between the positive expression of TIMP-1 and the invasiveness and metastasis was negative. The relationship of MMP-9 and TIMP-1 to the invasiveness and metastasis in gastric carcinomas was that MMP-9 promotes and TIMP-1 inhibits tumor metastasis. The patients with MMP-9 positive expression and TIMP-1 negative expression are poorer prognosis.

AIM: The expressive balance between matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor of metalloproteinase-1 (TIMP-1) plays a critical role in maintaining the degradation and synthesis of extracellular matrix. Loss of such balance is associated with invasion and metastasis of tumors. This study aimed to determine the expression of MMP-9 and TIMP-1 in gastric carcinoma, and the association of the expressive imbalance between MMP-9 and TIMP-1 with the invasion and metastasis and prognosis of gastric carcinoma. METHODS: We used immunohistochemistry to determine the expressions of MMP-9, TIMP-1 and proliferating cell nuclear antigen Ki-67 in the gastric specimens taken from 256 patients with primary gastric carcinoma. The patients were followed-up for up to 96 months. RESULTS: No association between the expression of MMP-9 and TIMP-1 and patients sex and age, tumor size and location of gastric carcinoma was observed. The incidence of the positive expression of MMP-9 in cases with tumors invasion to muscularis propria and visceral peritoneum (70.13 % and 69.09 %, respectively) was significantly higher than that in cases with tumor invasion only to lamina propria or submucosa (42.50 %, P=0.0162). The positive correlation between MMP-9 expression and the depth of tumor invasion was observed (Pearson correlation coefficient=0.2129, P=0.016). Along with the increase of the metastatic station of lymph nodes, the incidence of the MMP-9 expression was increased by degrees; a positive correlation between them was observed (Pearson correlation coefficient=0.2910, P=0.0001). There was also a significant correlation between MMP-9 expression and the TNM stage in gastric carcinoma (Pearson correlation coefficient=0.3027, P<0.0001). The incidence of MMP-9 expression in stage II and III/IV (75.00 % and 76.15 %, respectively) was significantly higher than those in stage I (46.15 %, P<0.0001). A negative correlation between TIMP-1 immunoreactivity and the depth of invasion, status of lymph node metastasis and TNM stage was observed (Pearson correlation coefficient =-0.1688, -0.3556 and -0.3004, P=0.023, <0.0001 and <0.0001, respectively). Four types of co-expression of MMP-9 and TIMP-1 were observed; i.e. MMP-9 positive but TIMP-1 negative (n=115), both positive (n=52), both negative (n=62) and MMP-9 negative but TIMP-1 positive (n=27). The frequency of serosal invasiveness was significant higher in patients with MMP-9 but without TIMP-1 expression than those with other types of the co-expression (P=0.0303). The incidence of lymph node metastasis was highest in patients with MMP-9 but without TIMP-1 expression, and lowest in those with TIMP-1 but without MMP-9 expression (P<0.0001). The survival rate in patients with MMP-9 but without TIMP-1 expression was lower than that in those with TIMP-1 but without MMP-9 expression (P=0.0014). CONCLUSION: Our results in gastric carcinoma demonstrated a significant positive association of MMP-9 over-expression with proliferation of tumor cells, the depth of invasiveness, lymph node metastasis and TNM stage, suggesting MMP-9 can serve as a molecular marker of tumor invasion and metastasis. We also demonstrate a significant negative relationship of TIMP-1 expression with the depth of invasiveness and lymph node metastasis, which provide a new idea in the tumor biological and genetic treatment. The interaction between MMP-9 and TIMP-1 in the processes of tumor invasion and metastasis is that MMP-9 mainly promotes tumor invasion and metastasis and TIMP-1 inhibits functions of MMP-9. The imbalance between MMP-9 and TIMP-1 expression may suggest the occurrence of tumor invasion and metastasis, predict poor prognosis. For patients with imbalanced MMP-9 and TIMP-1 expression, the optimal treatment scheme needs to be selected.

AIM: To investigate the relationship between the staging by endoscopic ultrasonography (EUS) and the expression of carcinoma metastasis associated gene in the patients with gastric carcinoma. METHODS: Sixty-three patients with gastric cancer were diagnosed by electric gastroscopy and EUS. The preoperative staging of gastric cancer was measured by EUS and compared with pathologic staging and MMP-9 expression. Peripheral serum level of MMP-9 was measured with enzyme-linked immunosorbent assay (ELISA), while the expression of MMP-9 protein was tested with immunohistochemistry and hybridization in situ in the gastric carcinoma tissues. RESULTS: The total accuracy of EUS in estimating invasive depth of gastric cancer was 80.95%, while that in estimating lymphatic metastasis was 73.02%. Serum MMP-9 levels were consistent with the expression of MMP-9 protein and MMP-9 mRNA in tissue, a result closely correlated with invasive degree, staging with EUS and lymphatic metastasis in gastric cancer (P<0.05). The total accuracy of estimating invasive depth in gastric cancer was 95.22% using both EUS and MMP-9. CONCLUSION: The MMP-9 level of preoperative serum presents the reference value for preoperative staging by EUS in the patients with gastric cancer. When serum MMP-9 level in gastric cancer is significantly high, physicians should pay closer attention to the metastasis which reaches the serosa or beyond. Combining EUS and MMP-9 improves the accuracy in deciding the invasion and metastasis in the patients with gastric carcinoma.

Degradation components of basement membrane could be crucial for tumor invasion. A key role in this process has been assigned to cysteine proteases, i.e. cathepsins and matrix metalloproteinases. The purpose of this study was to investigate the relationship of the expression of MMP-9 and cathepsin B with tumor aggressiveness expressed by lymph node metastases and survival rates in gastric carcinoma patients. Slides of 5 mum-thick serial sections from 91 patients with primary gastric carcinoma were prepared and analyzed for MMP-9 and cathepsin B expression using anti-human monoclonal antibody (NCL-MMP-9 clone; dilution 1:40 and NCL-CATH-B clone; dilution 1:40). The patients were clinically monitored for 84 months. We found no association between the expression of MMP-9 and cathepsin B in main mass of tumor and patients' gender, tumor location, Lauren's classification or histological differentiation. Also no correlation was observed between the expression of MMP-9 in main mass of tumor and depth of invasion. A strong statistically significant association was found between the expression of MMP-9 and cathepsin B in main mass of tumor and lymph node involvement (p<0.001; p<0.001, respectively). However, we observed no correlation between the expression of MMP-9 and cathepsin B in main mass of tumor and lymph node involvement or 5-year overall survival. Our results may suggest that the expression of matrix metalloproteinase-9 (MMP-9) and cathepsin B is correlated with lymph node metastasis in advanced gastric carcinoma, but not with patients' postoperative survival.

Approximately 10% of gastric carcinomas (GC) worldwide are associated with Epstein-Barr virus (EBV). GC is one of the most frequent human malignancies associated with EBV. The latent expression of the EBV-oncogene BARF1 is restricted to epithelial malignancies. To investigate the underlying BARF1-related mechanisms of oncogenic epithelial transformation, we analyzed gene expression profiles of a BARF1-transfected epithelial (HaCaT+) and the corresponding BARF1-negative (HaCaT-) cell line by cDNA microarray analysis. Real-time PCR was performed to confirm the cDNA microarray results. In addition, immunohistochemistry and fluorescence in situ hybridization were performed on a tissue microarray of 181 GC including 11 EBV-associated GC. Among other genes cyclin D1 expression was significantly upregulated in HaCaT+ on the transcriptional and protein level. Cyclin D1 protein expression in GC revealed a significant overexpression of cyclin D1 in EBV-associated GC (p<0.012) but not in EBV-negative GC. Cyclin D1 FISH showed that cyclin D1 overexpression was not due to gene amplification in EBV-associated GC. Cyclin D1 is induced in HaCaT+ by BARF1 and is overexpressed in EBV-associated GC indicating an interaction of viral BARF1 and cyclin D1.

PURPOSE: Like most cancers, gastric cancer has a complex multistep etiology that involves both environmental and genetic factors. Matrix metalloproteinase-9 (MMP-9) is frequently overexpressed in gastric cancer. We investigated the effect of the genetic differences in MMP-9 coding region on the occurrence and progression of gastric cancer. EXPERIMENTAL DESIGN: A case-control study was conducted in a population of 74 patients and 100 healthy people in southeast China. Individuals were genotyped for two single nucleotide polymorphisms (SNP) in MMP-9: R279Q and P574R. Genotypic distributions between patient and control groups were compared for correlations with cancer occurrence. Associations between genotypic distributions and several clinicopathologic features were also analyzed using univariate tests, multivariate logistic regression modeling, and stratified analyses. RESULTS: Significant associations were revealed between both SNPs and lymph node metastasis [P = 0.012 and 0.025; odds ratio (OR), 3.4 and 2.8, respectively]. After adjustment using logistic regression for the potential confounding effects of gender, age, and location of the tumors, homozygous MMP-9 279RR and 574PP are more evidently associated with lymph node metastasis with OR(adjusted) of 5.7 [95% confidence interval (95% CI), 1.80-18.34] and 4.2 (95% CI, 1.37-12.69). The homozygous 279R-574P haplotype showed a stronger association by an OR(adjusted) of 6.1 (95% CI, 1.92-12.29) and was also associated with the 1-year postoperative mortality (OR(adjusted), 6.5; 95% CI, 1.18-35.74). Interestingly, our data also suggested that the MMP-9 polymorphisms seem to result in higher risk of lymph node metastasis through a pathway independent of cancer invasion because no positive associations were found between these polymorphisms and cancer invasion (OR, 0.59 < 1). The stratified analyses indicated a synergistic interaction between the MMP-9 polymorphisms and the type of diffuse in affecting lymph node metastasis (OR, 13.4; P(between strata) = 0.04). Significant association between both SNPs and the overall occurrence of gastric cancer was not observed. CONCLUSION: The present study has shown significant associations between the two nonsynonymous MMP-9 polymorphisms with lymph node metastasis in gastric cancer, especially with the diffuse type. The relatively large values of ORs and disassociation with cancer invasion suggest that the genetic differences of MMP-9 protein play an important and specific role in lymph node metastases, and therefore, further investigation of the underlying molecular mechanism is warranted.

Matrix metalloproteinases (MMPs) play an important role in the extracellular matrix degradation, that is an essential step in tumor invasion and metastases. The current study objective was to evaluate the expression of MMP-9 in the neoplastic and in the interstitial inflammatory infiltrate cells in gastric cancer (GC). Moreover, the relationship between expression of this enzyme and clinicopathological features of GC, such as TNM stage, the depth of tumor invasion, lymph node and distant metastases were assessed. The study comprised 54 patients with gastric cancer. Immunohistochemistry was used to determine the expression of MMP-9 in gastric cancer cells. The semi-quantitative scale was applied to evaluate the expression of metalloproteinase-9. Immunohistochemical testing revealed a positive reaction of MMP-9 in 98% of all cancer tissue specimens and in 93% of inflammatory cells. The expression of MMP-9 in the neoplastic and inflammatory cells increased with more advance tumor stage, depth of tumor invasion and presence of lymph node as well as distant metastases. These findings indicate the significance of interstitial inflammatory infiltrate cells in the MMP-9 synthesis and the role of this enzyme in the invasiveness and metastatic potential of GC.

AIM: to investigate the expression of integrinalphanubeta6 and MMP-9 in gastric cancer and the correlation with clinicopathologic characteristic. METHODS: the expression of integrinalphanubeta6 and MMP-9 protein was detected with immunohistochemical EnVision plus non-hiotin technique in 94 cases of gastric cancer and pericarcinoma tissues. The correlation with clinicopathologic characteristic were analyzed. RESULTS: the positive expression rates of integrinalphanubeta6 and MMP-9 protein in gastric cancer were significantly higher that tat in pericarcinoma tissues (P<0.01). The expression of integrinalphanubeta6 were positively related to Lauren type, tumor clinical stage, differentiation degree and lymph node metascasis (N stage) (P<0.01). The expression of MMP-9 were positively related Lauren type, tumor clinical stage, differentiation degree, depth of invasion and lymph node metascasis (N stage)(P<0.01). The expression of integrinalphanubeta6 was positively correlated with MMP-9 (r=0.672, P<0.01). Trough Cox multivariate regression analysis, Lauren type, lymph node metascasis (N stage), the expression of integrinalphanubeta6 and MMP-9 showed significant associations with 5-year survival rate. CONCLUSION: the overexpression of integrinalphanubeta6 and MMP-9 in gastric cancer is closely correlated with tumor invasion and metastasis. They may be as prognostic markers and target proteins.

Secretory leukocyte protease inhibitor (SLPI) protects tissue from proteases, and promotes cell proliferation and healing during inflammatory response. SLPI is also overexpressed in gastric, lung and ovarian cancers, which accelerates the metastasis of cancer cells. Matrix metalloproteinases-2, -9 (MMP-2 and MMP-9) are overexpressed in high metastatic cancers, and promote the migration of cancer cells through collagen degradation. SLPI and MMP-2, -9 are critical factors in stimulating the metastatic processes but there are no reports of a direct correlation between these molecules. Therefore, this study examined the role of SLPI related to MMP-2 and MMP-9 using two gastric cancer cell lines, such as characterized non-metastatic SNU484 and highly metastatic SNU638 cells. SLPI, MMP-2 and MMP-9 mRNA and protein expression were higher in SNU638 cells than in SNU484 cells. In addition, the rate of cell migration and invasion was higher in the SNU638 cells than in SNU484 cells. Interestingly, after treatment with SLPI, the rate of migration and invasion was higher in the SNU484 cells than in the positive control (PC) SNU484 cells. The rate of migration was also higher in the SNU638 cells after SLPI treatment than in the SNU638 cells (PC) but the invasion rate was not changed. The expression and secretion of MMP-2 and MMP-9 as well the rate of cell migration and invasion were significantly lower in SLPI-siRNA transfected SNU638 cells (si-SLPI/SNU638) but higher in SLPI-treated SNU484 cells (SNU484 + SLPI). Strong Elk-1 phosphorylation was detected in SNU484 + SLPI and SNU638 cells but was barely detectable in SNU484 and si-SLPI/SNU638 cells. These results show that SLPI promotes the metastasis of SNU638 gastric cancer cells by increasing MMP-2 and MMP-9 expression through Elk-1 signaling, indicating its role as a signaling molecule not a protease inhibitor.

Activation of sonic hedgehog (Shh) signaling has been implicated in progression of a variety of tumors. In this study, we elucidated a role for Shh in the invasion of gastric tumors and determined the mechanism by which Shh is regulated. Immunohistochemical analysis of 178 primary human gastric tumor biopsies indicated that Shh expression was positively correlated with lymph node metastasis, high lymphatic vessel density, and poor prognosis. In mouse xenograft models of human gastric cancer, enforced expression of Shh significantly enhanced the incidence of lung metastasis compared with nonexpressing controls. Mechanistic investigations revealed that phosphoinositide 3-kinase (PI3K)/Akt inhibition blocked Shh-induced epithelial-mesenchyme transition, the activity of matrix metalloproteinase 9 (MMP-9), and lymphangiogenesis, reducing tumor invasiveness and metastasis. Taken together, our findings establish that Shh signaling promotes the metastasis of gastric cancer through activation of the PI3K/Akt pathway, which leads to mesenchymal transition and MMP-9 activation. These findings offer preclinical validation of Shh as a candidate therapeutic target for treatment of metastatic gastric cancers.

BACKGROUND/AIMS: Matrix metallopeptidase (MMP) is known to be involved in tumor invasion and metastasis of cancer. This study investigated the association of MMP7 rs11568818, MMP8 rs11225395, MMP9 rs17576 and rs2250889 with gastric cancer (GC) development and lymph node metastasis (LNM). METHODS: Samples were obtained from 326 chronic gastritis (CG) and 153 GC patients and genotyped by using the GoldenGate(R) method. Chi-square test was performed to identify the difference of allele distribution between each group (CG vs. GC; CG vs. with LNM GC). The associations of genotype with risk of GC and LNM were estimated by odds ratio and the 95% confidence interval was calculated by logistic regression adjusting for age and sex. RESULTS: The allele and genotype frequencies of MMP7 rs11568818, MMP8 rs11225395, MMP9 rs17576 and rs2250889 were not associated with the development of GC and LNM. CONCLUSIONS: In summary, MMP7 rs11568818, MMP8 rs11225395 MMP9 rs17576 and rs2250889 were not associated with the GC development and LNM in Korean population.

Semaphorin 5A, a member of semaphorin family, was originally identified as axonal guidance factor functioning during neuronal development. Previously, we showed that the expression of semaphorin 5A might contribute to the metastasis of gastric cancer. However, its functional roles and mechanism(s) in invasion and metastasis of gastric cancer remain unclear. By using human gastric caner cell lines Parental SGC7901, SGC7901-siScrambled and SGC7901-siSema 5A, we found that semaphorin 5A significantly promoted the invasive and metastatic abilities of gastric cancer cell in vitro. Semaphorin 5A increased the expression of MMP9 by activating phosphorylated ErK1/2 in gastric cancer cell. Furthermore, MEK inhibitor PD98059 and MMP9 antibody (Ab) significantly inhibited in vitro invasive and metastatic abilities induced by semaphorin 5A. Taken together, the present work revealed a novel function of semaphorin 5A that the existence of semaphorin 5A could promote invasion and metastasis of gastric cancer by regulating MMP9 expression, at least partially, via the MEK/ERKs signal transduction pathway. Semaphorin 5A and its regulated molecules could be the potential targets for cancer therapy.

BACKGROUND: Coronins are a family of highly evolutionary conserved proteins reportedly involved in the regulation of actin cytoskeletal dynamics, although only coronin 3 has been shown to be related to cancer cell migration. In glioblastoma cells, the knockdown of coronin 3 inhibits cell proliferation and invasion. Coronin 3 is also associated with the aggression and metastasis of hepatocellular carcinoma. In this paper, we analyze the migration, invasion and metastasis abilities of gastric cancer cells after up- or down-regulation of coronin 3, and explore the mechanism of coronin 3 in the process of gastric cancer metastasis. RESULTS: The expression of coronin 3 was higher in the highly metastatic sub-cell line MKN28-M, which we established in our laboratory. We also demonstrated that the expression of coronin 3 was remarkably higher in lymph lode metastases than in primary gastric cancer tissues, and over-expression of coronin 3 was correlated with the increased clinical stage and lymph lode metastasis. Recombinant lentiviral vectors encoding shRNAs were designed to down-regulate coronin 3 expression in gastric cancer cell lines. Stable knockdown of coronin 3 by this lentiviral vector could efficiently inhibit the migration and invasion of MKN45 gastric cancer cells. In contrast, up-regulation of coronin 3 significantly enhanced migration and invasion of MKN28-NM cells. In addition, knockdown of coronin 3 significantly reduced liver metastasis in mice after tail vein injection of gastric cancer cells. The Human tumor metastasis PCR Array was used to screen the metastasis-associated genes identified by the down-regulation of coronin 3, and the results suggested that, following the knockdown of coronin 3, the tumor cell migration and invasion were inhibited by the reduced expression of MMP-9 and cathepsin K. CONCLUSION: Coronin 3 is highly expressed in gastric cancer metastases and can promote the metastatic behaviors of gastric cancer cells, including their migration and invasion.

BACKGROUND: Interleukin-1beta (IL-1beta) has been implicated in the progression of gastric adenocarcinoma (GA); however, the molecular mechanisms of action of IL-1beta in GA are poorly characterized. P38 and JNK are the major MAPK family members that regulate IL-1beta signaling pathways. Here, we investigated the role of both p38 and JNK in IL-1beta-induced GA cell migration, invasion and metastatic potential. METHODS: The effects of IL-1beta-induced p38 and JNK activation in GA cells were determined using in vitro Transwell migration and invasion assays of MKN-45 and AGS cells, or an in vivo metastasis assay in nude mice. The IL-1beta-induced p38 signaling pathway was further characterized in GA cells. Activation of the IL-1beta/p38 signaling pathway was also assessed in human primary GA tissues by immunohistochemistry. RESULTS: IL-1beta-induced activation of p38 increased GA cell migration and invasion in vitro and promoted the metastatic potential of GA cells in vivo; these effects were attenuated by p38 siRNA or the p38 inhibitor SB202190. MMP2 or MMP9 siRNAs and the MMP2/9 inhibitor BiPS also inhibited IL-1beta-induced GA cell migration and invasion in vitro. IL-1beta-induced p38 activation significantly increased MMP2 and MMP9 mRNA and protein expression and activity. Luciferase reporter assays demonstrated that the activator protein-1 (AP-1) and the AP-1 binding sites of the MMP9 promoter (-670/MMP9) were activated by IL-1beta-induced p38 activation. Phospho-p38 was significantly upregulated in human GA tissues (compared to matched non-neoplastic tissues), and significantly associated with lymph node metastasis, and invasion beyond the serosa. expression of phospho-p38 significantly correlated with IL-1beta, MMP2, MMP9, and c-fos expression in both human GA tissues and GA cell metastases in the lungs of nude mice. IL-1beta was also capable of activating JNK in GA cells, but activation of JNK was not associated with GA cell migration and invasion. Therefore, IL-1beta-induced the migration and invasion in GA cells were regulated by p38, but not by JNK. CONCLUSIONS: IL-1beta-induced p38 activation and the IL-1beta/p38/AP-1(c-fos)/MMP2 & MMP9 pathway play an important role in metastasis in GA; this pathway may provide a novel therapeutic target for GA.

The levels of metalloproteinases (MMP-2,-9), their tissue inhibitors (TIMP-1,-2) and extracellular matrix metalloproteinase inducer (EMMPRIN) were studied in tumor tissue and blood serum from patients with head and neck squamous sell carcinoma. Immunohistochemical investigation showed much higher expression of MMP-9 and TIMP-1 in tumor tissue than MMP-2 and TIMP-2. Different expression of the studied parameters (except TIMP-1) in cancer cells and stroma was observed. Enhanced level of MMP-2,-9 and TIMP-2 expression was revealed mainly in fibrocytes, leukocytes and stromal extracellular matrix. expression of EMMPRIN in tumor cells was higher than in stromal cells. It is possible that carcinoma cells express EMMPRIN to increase MMP production by surrounding cells. Significant decrease of inhibitor expression was observed in cells of carcinomas with N1 grade of metastasis than in tumors without metastasis. Also the decrease in TIMP-1 level was determined in blood serum of patients with metastasis to regional lymph nodes compared with serum of patients without metastasis. Thus, MMP-9 and TIMP-1 play an important role in the development of head and neck squamous sell carcinoma and TIMP-1 level in blood serum and cancer tissues is linked to first grade of regional lymph node metastasis.

The mechanisms leading to squamous cell carcinoma of head and neck (SCCHN) metastasis are not fully understood. Although evidence shows that the chemokine receptor 7 (CCR7) and its ligand CCL19 may regulate tumor dissemination, their role is not clearly defined in SCCHN. Matrix metalloproteinases break consisting of tissue barrier to the surrounding tissue invasion and metastasis by destroying the balance of matrix degradation of the basement membrane of tumor cells and extracellular matrix (ECM). We used chemotaxis and migration assays, western blotting, gelatin zymography, actin polymerization assay, immunofluorescence staining and immunohistochemical analysis to explore whether MMP-9 can be activated by CCL19 (CCR7's ligand) and its role in SCCHN. The experiments were performed in the metastatic SCCHN cell line PCI-37B after pre-incubation of the cells with CCL19 and SB-3CT (inhibitor of MMP-9). Our results demonstrated that CCR7 favors PCI-37B cell chemotaxis and migration, upregulation of MMP-9 protein and motivates the activity of MMP-9 protein, induces reorganization of the actin cytoskeleton and upregulation of MMP-9 protein. SB-3CT can block all these effects. Collectively, our data indicated that CCR7 regulates cell chemotaxis and migration via MMP-9 in metastatic SCCHN, and these results provide a basis for new strategies in preventing metastases of SCCHN.

OBJECTIVE: To evaluate the expression of endothelin-1(ET-1) and matrix metalloproteinase-9 (MMP-9) in laryngeal carcinoma and their correlations with clinical features. METHOD: The expression of ET-1 and MMP-9 was detected by immunohistochemical method in 58 specimens of laryngeal carcinoma, 28 specimens of polyps of vocal cord and 19 specimens of normal laryngeal tissues. RESULT: The expressions of ET-1, MMP-9 in laryngeal carcinoma were remarkably higher,compared to polyps of vocal cord and normal laryngeal tissues (P < 0.05). The expression of ET-1, MMP-9 was associated with clinical stage, T stage and lymph node metastasis. expression levels of ET-land MMP-9 correlated significantly with each other (r(s) = 0.693, P < 0.05). CONCLUSION: The expression of ET-1 and MMP-9 may be the vital indexes in laryngeal carcinoma.

OBJECTIVE: To investigate the expression and clinical significance of OPN, CD44v6 and MMP-9 in laryngeal squamous cell carcinomas (LSCC). METHOD: The expression of OPN, CD44v6 and MMP-9 in the 47 cases of LSCC and 10 cases normal mucosa were detected by immunohistochemical method. The correlation between OPN, CD44v6 and MMP-9 proteins expressions and the correlation between them and clinicopathological feature were also studied. RESULT: (1) The positive expression rates of OPN, CD44v6 and MMP-9 were 63.8%, 76.6% and 68.1% in LSCC of 47 cases and, and 10%, 30% and 0 in the tumor adjacent issue. (2) The positive rate of OPN and CD44v6 was significantly correlated with the clinical staging, the pathological grading and the metastasis of cervical lymph node (P < 0.05). The positive rate of MMP-9 was correlated with the clinical staging and the metastasis of cervical lymph node (P < 0.05). The positive rates of OPN, CD44v6 and MMP-9 were not correlated with patient's age, sex and tumor location (P > 0.05). (3) The expression of OPN was well correlated with the positive rate of CD44v6 and MMP-9 in LSCC (r = 0.421, P < 0.01, r = 0.340, P < 0.05). The expression of positive rate of CD44v6 and MMP-9 was well related in LSCC as well (r = 0.376, P < 0.01). CONCLUSION: There is a high level of expression of OPN, CD44v6 and MMP-9 in LSCC. OPN, CD44v6 and MMP-9 are positively related and involved in the invasion and metastasis of LSCC.

Des-gamma-carboxy prothrombin (DCP), an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells, has been shown to be associated with the biological malignant potential of HCC. The aim of this study was to evaluate the effect of DCP on HCC cell growth and metastasis, and to explore the underlying molecular mechanisms. DCP significantly stimulated HCC cell growth, as measured by cell counting kit-8 assay. Transwell chamber assay showed that DCP increased HCC cell migration through reconstituted extracellular matrix (Matrigel). Gelatin zymography assay and Western blot analysis demonstrated that DCP increased the secretion and expression of matrix metalloproteinase (MMP)-2 and MMP-9 in the supernatant of cultured HCC cells and on tumour cell membranes. DCP was found to bind to the cell surface receptor Met, resulting in Met phosphorylation and subsequent activation of the epidermal growth factor receptor (EGFR). Western blot analysis demonstrated that DCP stimulated a sequential kinase phosphorylation cascade including ERK1/2, MEK1/2 and c-Raf, indicating activation of the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK1/2 MAPK) signalling pathway. Furthermore, blocking ERK1/2 MAPK activation with ERK1/2 inhibitor PD98059 essentially abolished the DCP-induced MMP-2 and MMP-9 activity, confirming the signalling pathway of DCP stimulation. Taken together, these results suggested that DCP stimulates HCC growth and promotes HCC metastasis by increasing the activity of MMP-2 and MMP-9 through activation of the ERK1/2 MAPK signalling pathway.

Cyclophilin A (CypA) is a member of peptidyl prolyl isomerases (PPIases), which catalyze the cis/trans isomerization of prolyl peptide bonds on the NH-terminal side of Pro residues in peptide chains. Altered expression of CypA has been reported in hepatocellular carcinoma (HCC), but the biological functions of CypA in HCC remain unknown. We found that the level of CypA expression correlated with the metastatic capability of two HCC cell lines, MHCC97-L and MHCC97-H. Stable expression of ectopic CypA in SK-Hep1 cells promotes cell adhesion, motility, chemotaxis, and in vivo lung metastasis, without affecting cell proliferation. We further analyzed microarray results to identify target genes controlled by CypA. Twenty-one genes related to metastasis were altered by CypA over-expression. A member of matrix metalloproteinase, MMP3, was identified by microarray analysis. The regulation of MMP3 and its homologue MMP9 by CypA were further confirmed by quantitative real-time RT-PCR and zymography assay. Taken together, our data suggest that CypA promotes HCC cell metastasis at least partially through up-regulation of MMP3 and MMP9.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide; the prognosis of HCC patient remains poor owing to intrahepatic and extrahepatic metastasis and postsurgical recurrence. The aim of the present study is to determine the molecular mechanisms underlying the metastasis of HCC. METHODS: HCC patients and treated HCC cells were molecularly characterized by miRNA microarray analysis, qRT-PCR, Western blots, transwell assay, and immunohistochemistry. RESULTS: Here, by employing a miRNAs microarray analysis, we found that miR-491 level was the most significant down-regulation in poorly differentiated HCC tissue compared with well-differentiated HCC tissue. We then selected HepG2 (very low migratory capacity), MHCC97L (low migratory capacity) and MHCC97H (high migratory capacity), as well as HCC tissues with different status to further investigate the effects of miR-491 on the metastasis of HCC. Our data showed that miR-491 levels were inversely correlated with different status of differentiation in HCC tissues and with migratory potential in HCC cell lines. In HepG2 cells, inhibition of miR-491 increased the expression of matrix metalloproteinase 2/9 (MMP-2/9) and the migratory potential; however, in MHCC97H cells, overexpression of miR-491 level decreased the expression of MMP-2/9 and the migratory capacity. Moreover, miR-491 had a positive relationship with E-cadherin level; however, it had a negative relationship with vimentin level both in cell lines and tissue samples of HCC. MiR-491 levels of non-metastasis HCC tissue are higher than that of metastasis HCC tissue. CONCLUSION: Our results suggest that miR-491 is involved in metastasis of HCC by blocking EMT and decreasing MMP-9 levels, which may provide a new clue for preventing tumour metastasis of HCC.

BACKGROUND AND OBJECTIVES: We previously showed that the calcium-binding protein S100A4 is overexpressed and related to metastasis in hepatocellular carcinoma (HCC). However, whether S100A4 participates in the regulation of metastasis and its mechanisms in HCC is mostly unknown. Given the associations of S100A4, nuclear factor-kB (NF-kB/RelA) and MMP-9 with metastasis in a variety of malignancies, we explored a potential role of S100A4 in HCC metastasis and its mechanism. METHODS: 20 patients with HCC invasion (Lymph node metastasis, microvascular invasion, major portal vein invasion and intrahepatic metastasis) and 20 patients without HCC invasion were included. These tissues were detected for the expression of S100A4, NF-kB/RelA and MMP-9 by immunohistochemistry and quantitative real time polymerase chain reaction (Q-PCR). Correlation between the expressions of S100A4, NF-kB/RelA and MMP-9 with the invasion was analysed. The expressions of S100A4, nuclear factor-kB and MMP-9 was evaluated in HepG2 cells by western blot and immunohistochemistry. HepG2 cells were stably transfected with S100A4-specific small interfering RNA (S100A4 siRNA) to knockdown of S100A4, then transiently transfected with S100A4 cDNA to rescure the S100A4 level and evaluated for effects on invasion and expression analysis for molecules involved in invasion. After the HepG2 cells recurred the S100A4 levels, the HepG2 cells was treated with 5 microM Pyrrolidine Dithiocarbamate (PDTC) (a selective NF kappa B inhibitor) to inhibit the NF-kB activity, or treated with Batimast (BB94: a MMPs inhibitor) to inhibit the MMP-9 activity. The expression analysis for molecules involved in invasion was analyzed. RESULTS: A significant increase of S100A4, NF-kB/RelA and MMP-9 expression in HCC tissues with invasion than that of without invasion. A positive correlation was observed between S100A4, NF-kB/RelA, MMP-9 and invasion, respectively. In addition, S100A4 was positively correlated with NF-kB and MMP-9. S100A4 siRNA mediated knockdown of S100A4 in HepG2 cells resulted in significant reduction in the NF-kB activity and MMP-9 expression, and dramatically decreased its invasion. Moreover, the HepG2 cell metastatic potential was rescued by overexpression of S100A4 completely, at the same time, the NF-kB activity and MMP-9 expression was also increased. Pretreatment with PDTC or BB94 was observed to significantly reduce NF-kB activity and MMP-9 expression and dramatically decreased S100A4 -induced invasion. CONCLUSIONS: Our findings indicate that S100A4 contributes to HCC metastasis by activation of NF-kB dependent MMP-9 expression, suggesting S100A4 as a novel diagnostic biomarker and therapeutic target in HCC.

PURPOSE: Matrix metalloproteinase 9 (MMP-9) plays critical roles in cancer development and aggression. Nonsynonymous single-nucleotide polymorphisms (SNP) in the functional domain of the MMP-9 gene may influence substrate and inhibitor binding and contribute to cancer predisposition and aggression. PATIENTS AND METHODS: To test our hypothesis that common nonsynonymous SNPs, R279Q, P574R, and R668Q, in MMP-9 are associated with lung cancer development and metastasis, we conducted a case-control study of 744 patients with incident lung cancer and 747 cancer-free controls in Southeast China. Multivariate logistic regression analysis was used to calculate adjusted odds ratio (OR) and 95% confidence interval (95% CI). RESULTS: We found that compared with the 279QQ genotype, the 279RR genotype was associated with significant elevated risk of lung cancer with metastasis (adjusted OR, 1.79; 95% CI, 1.03-3.08), whereas the 574PR heterozygote and 574PP homozygote had 1.46-fold (95% CI, 0.94-2.26) and 1.69-fold elevated risk (95% CI, 1.10-2.60), respectively, compared with the 574RR genotype. When we examined the combined effect of R279Q and P574R and used the 279R and 574P as the risk alleles, a significantly increased risk of lung cancer was associated with both the genotypes containing "1 to 2 risk alleles" (adjusted OR, 2.16; 95% CI, 1.30-3.59) and containing ">2 risk alleles" (adjusted OR, 2.44; 95% CI, 1.48-4.03), and it was more pronounced in 290 lung cancer cases with metastasis [adjusted OR, 2.30 (95% CI, 1.09-4.85) for the 1 to 2 risk alleles subgroup and adjusted OR, 2.82 (95% CI, 1.35-5.88) for the >2 risk alleles subgroup], compared with those without any risk alleles. However, no overall significant associations were observed between R668Q and lung cancer risk in this study population. CONCLUSION: These findings indicate that the potentially functional polymorphisms, MMP-9 P574R and R279Q, may confer the biomarker in the occurrence and metastasis of primary lung cancer. Further functional studies including these two genetic variants are warranted to confirm our findings.

The role of matrix metalloproteinase (MMP)9 in lung cancer progression is controversial. MMP9 promotes local tumor progression and distant metastasis in mouse models by enhancing extracellular matrix degradation, releasing VEGF from extracellular matrix and promoting vascular pericyte recruitment. Furthermore, increased plasma MMP9 expression levels in human subjects with metastatic non-small cell lung cancer (NSCLC) inversely correlates with survival. In contrast, MMP9 can benefit the host by generating inhibitors of endothelial cell proliferation such as angiostatin and NC1 domains of collagen IV. To better understand the role of host MMP9 on the primary growth and metastatic potential of NSCLC, we performed an orthotopic model of NSLC in integrin alpha1-null mice (a genetic model for increased MMP9). In these mice we observed decreased number, size and vascularization of primary NSCLC tumors when compared to wild type controls. In addition, decreased number and size of NSCLC-derived metastases were evident in the alpha1-null mice. Furthermore, pharmacological inhibition of MMPs in the alpha1-null mice at the time of tumor cell injection resulted in an increase in the number of both primary and metastatic lung cancer as compared to untreated mice, suggesting that primary growth and metastases of NSCLC are worsened by the early inhibition of MMPs. In conclusion, although MMP9 may potentially promote tumor growth and metastasis, production of MMP-dependent anti-angiogenic factors seems to override these effects and protects the host from NSCL growth and progression.

RECK is a membrane-anchored glycoprotein which may negatively regulate matrix metalloproteinase (MMP) activity to suppress tumor invasion and metastasis. In this study, recombinant proteins corresponding to the residues 285-368 (named as CKM which contained cysteine knot motif), 605-799 (named as K123 which contained three Kazal motifs), 676-799 (named as K23 which contained the last two Kazal motifs) and full-length RECK were produced and their anti-cancer effects were tested. Full-length RECK and K23 but not K123 and CKM inhibited MMP9 secretion and activity. In addition, RECK and K23 inhibited invasion but not migration of metastatic lung cancer cells in vitro. Protein binding and kinetic study indicated that K23 physically interacted with MMP-9 and inhibited its activity by a non-competitive manner. Moreover, K23 reduced metastatic tumor growth in lungs of nude mice. Taken together, our results suggest that the K23 motifs of RECK protein can inhibit MMP-9 secretion and activity and attenuate metastasis of lung cancer cells.

It is well known that matrix metalloproteinases (MMPs) act an important role in the invasion, metastasis and angiogenesis of cancer cells. Agents suppressed the MMPs could inhibited the cancer cells migration and invasion. Numerous evidences had shown that curcumin (the active constituent of the dietary spice turmeric) has potential for the prevention and therapy of cancer. Curcumin can inhibit the formation of tumors in animal models of carcinogenesis and act on a variety of molecular targets involved in cancer development. There is however, no available information to address the effects of curcumin on migration and invasion of human lung cancer cells. The anti-tumor invasion and migration effects of lung cancer cells induced by curcumin were examined. Here, we report that curcumin suppressed the migration and invasion of human non-small cell lung cancer cells (A549) in vitro. Our findings suggest that curcumin has anti-metastatic potential by decreasing invasiveness of cancer cells. Moreover, this action was involved in the MEKK3, p-ERK signaling pathways resulting in inhibition of MMP-2 and -9 in human lung cancer A549 cells. Overall, the above data shows that the anticancer effect of curcumin is also exist for the inhibition of migration and invasion in lung cancer cells.

CONTEXT: Mere15 is a novel antitumor polypeptide purified from Meretrix meretrix Linn. (Veneridae). Previous studies have shown that the polypeptide induced cell death via intrinsic mitochondrial pathway. OBJECTIVE: In the present study, the effects of Mere15 on cell adhesion, migration, invasion, as well as secretion and expression of matrix metalloproteinases (MMPs) were studied in human lung adenocarcinoma A549 cells. MATERIALS AND METHODS: The effect of Mere15 on cell adhesion, migration and invasion were studied by cell adhesion and transwell assay. The expression of MMPs was determined by gelatin zymography and RT-PCR analysis. RESULTS: The ability of cell adhesion was decreased by 86.74% at the concentration of 12.0 mug/mL of Mere15. And the migration and invasion of A549 cells were decreased with the inhibition ratio of, 69.22 and 53.84% when treated with 15.0 mug/mL of Mere15. Further study also revealed that treatment of the cancer cells with Mere15 (15.0 mug/mL), the secretion of MMP-2 and MMP-9 were down-regulated with the inhibition ratio of 72.00 and 93.24% and the inhibition rate of mRNA expression of MMP-2 and MMP-9 was 57.54 and 91.22%, respectively. DISCUSSION AND CONCLUSION: The study demonstrates that Mere15 inhibits tumor growth via both pro-apoptotic and antimetastasis pathways, and the polypeptide has potential to be developed as a multi-target therapeutic agent for the treatment of human lung cancer.

Immunohistochemical studies revealed increased level of matrix metalloproteinase 9 in skin melanoma cells. Inhibition of matrix metalloproteinase 9 with interfering RNA changed the level of PCNA and reduced N-cadherin content in melanoma cells. This attests to the involvement of matrix metalloproteinase 9 in the realization of invasion and metastatic growth and in the regulation of tumor growth and progress.

OBJECTIVE: To investigate the expression and significance of matrix metalloproteinases (MMP-2, MMP-9) and tissue inhibitor of matrix metalloproteinase (TIMP-2, TIMP-1) in non-melanoma skin cancer (NMSC). METHODS: Thirty six patients with squamous cell carcinoma (SCC) and 32 patients with basal cell carcinoma (BCC), confirmed by pathology, were selected, and 30 cases of normal skin were selected as control. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in all samples were examined by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The expression rate, expression intensity and expression level of each factor were recorded. The results were compared between the groups. RESULTS: The expression rates of MMP-2 and MMP-9 in the control group were 30.0% and 36.7%, the expression levels of MMP-2 and MMP-9 in the control group were 57.216 +/- 12.785 and 59.318 +/- 13.262, all significantly lower than those in the tumor edge and center of the SCC and BCC groups (P < 0.01). The expression rates of TIMP-1 and TIMP-2 in the control group were 96.7% and 100%, their expression levels were 121.738 +/- 25.516 and 122.612 +/- 25.964, all significantly higher than those in the SCC and BCC groups (P < 0.01). The expression levels of MMP-2 and MMP-9 in the tumor center and edge of SCC group were significantly higher than those in the corresponding parts of the BCC group, while the expression levels of TIMP-1 and TIMP-2 were significantly lower than those in the BCC group (P < 0.01). The expression levels of MMP-2 and MMP-9 in the tumor edge of the SCC and BCC groups were significantly higher than those in the tumor centers (P < 0.01), while the expression levels of TIMP-1and TIMP-2 were significantly lower than those in the tumor centers (P < 0.01). CONCLUSION: MMP-2, MMP-9 and TIMP-2, TIMP-1 may play an important role in the development, progression, invasion and metastasis of non-melanoma skin cancer.

The molecular mechanism of tissue-specific metastasis in tumors endogenously expressing members of the vascular endothelial growth factor (VEGF) family is not yet clear. Here we demonstrate that MMP9 is specifically induced in premetastatic lung endothelial cells and macrophages by distant primary tumors via VEGFR-1/Flt-1 tyrosine kinase (TK) and that it significantly promotes lung metastasis. In a genetic approach using mice, suppression of MMP9 induction by deletion of either VEGFR-1TK or MMP9 markedly reduced lung metastasis. Furthermore, the MMP9 levels in endothelial cells of normal lung lobes from patients carrying distant tumors were significantly elevated as compared with those from patients without tumors. Thus, a block of MMP9 induction via VEGFR-1 inhibition could be useful for the prevention of tumor metastasis in lung.

Gelatinase B (MMP-9) and galectin-3 are widely known to participate in tumor cell invasion and metastasis. Glycans derived from MMP-9 expressed in MCF-7 breast cancer and THP-1 myeloid leukemia cells were compared with those from MMP-9 expressed in natural neutrophils. The many O-linked glycans of neutrophil gelatinase B presented a cluster of mainly galactosylated core II structures, 46% of which were ligands for galectin-3; 11% contained two to three N-acetyllactosamine repeating units that are high-affinity ligands for the lectin. The glycan epitopes thus provide MMP-9 with both high-affinity and (presumably) high-avidity interactions with galectin-3. In contrast, the O-glycans released from MMP-9 expressed in MCF-7 and THP-1 cells were predominantly sialylated core I structures. Only 10% of MCF-7 and THP-1 gelatinase B O-glycans were ligands for galectin-3 and contained only a maximum single N-acetyllactosamine repeat. Consistent with the glycan analysis, surface plasmon resonance binding assays indicated that the cancer-associated glycoforms of MMP-9 bound galectin-3 with an affinity and avidity significantly reduced compared with those of the natural neutrophil MMP-9. Galectin-3 exists as a multimer that also binds laminin, providing a means of localizing neutrophil MMP-9 in the extracellular matrix (ECM). The analytical data presented here suggest that MMP-9 glycoforms secreted by tumor cells are unlikely to be tethered at the site of secretion, thus promoting more extensive cleavage of the ECM and providing a rationale for the contribution that gelatinase B makes to cancer cell metastasis.

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.

Cyclooxygenase (COX)-2 is known to correlate with poor cancer prognosis and to contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal prostaglandin E(2) (PGE(2))-prostaglandin E2 receptor (EP)3 signaling appears critical for tumor-associated angiogenesis and tumor growth. Here we tested whether the EP3 receptor has a critical role in tumor metastasis. Lewis lung carcinoma (LLC) cells were intravenously injected into WT mice and mice treated with the COX-2 inhibitor NS-398. The nonselective COX inhibitor aspirin reduced lung metastasis, but the COX-1 inhibitor SC560 did not. The expression of matrix metalloproteinases (MMP)-9 and vascular endothelial growth factor (VEGF)-A was suppressed in NS-398-treated mice compared with PBS-treated mice. Lungs containing LLC colonies were markedly reduced in EP3 receptor knockout (EP3(-/-)) mice compared with WT mice. The expression of MMP-9 and VEGF-A was downregulated in metastatic lungs of EP3(-/-) mice. An immunohistochemical study revealed that MMP-9-expressing endothelial cells were markedly reduced in EP3(-/-) mice compared with WT mice. When HUVEC were treated with agonists for EP1, EP2, EP3, or EP4, only the EP3 agonist enhanced MMP-9 expression. These results suggested that EP3 receptor signaling on endothelial cells is essential for the MMP-9 upregulation that enhances tumor metastasis and angiogenesis. An EP3 receptor antagonist may be useful to protect against tumor metastasis.

Two sulfated triterpene glycosides, holothurin A(1) (HA(1)) and 24-dehydroechinoside A (DHEA), isolated from the sea cucumber Pearsonothuria graeffei, are of the holostane type with 18(20)-lactone and identical carbohydrate chains. DHEA has a side chain 23 (24)-double bond, while HA(1) has a hydroxyl group at C-21. In this study, we compared the effects of DHEA and HA(1) on metastasis in vitro and in vivo. The results show that HA(1) and DHEA treatment significantly suppressed adhesion of human hepatocellular liver carcinoma cells (HepG2) to both matrigel and human endothelial cells (ECV-304) and inhibited HepG2 cell migration and invasion in a dose-dependant manner. HA(1) and DHEA reduced tube formation of ECV-304 cells on the matrigel in vitro and attenuated neovascularization in the chick embryo using the chorioallantoic membrane (CAM) assay in vivo. Immunocytochemistry analyses revealed that both HA(1) and DHEA significantly decreased the expression of the matrix metallo-proteinase-9 (MMP-9) and increased the expression level of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important regulator of MMP-9 activation. Western blot analyses demonstrated that HA(1) and DHEA remarkably abolished the expression of vascular endothelial growth factor (VEGF). The expression of nuclear factor-kappa B (NF-kappaB) was significantly decreased by HA(1), while DHEA treatment had no effect on the down regulation of NF-kappaB expression. These data suggest that both DHEA and HA(1) exert significant antimetastatic activities by inhibiting MMP-9 and VEGF expression. DHEA-induced antimetastasis was more potent than HA(1). In addition, only HA(1) treatment downregulated the expression level of NF-kappaB, suggesting that the antimetastatic activity of triterpene glycosides derived from P. graeffei can be either NF-kappaB-dependent or -independent, depending on their structure.

OBJECTIVE: To investigate the expressions of matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) in nasopharyngeal carcinoma and their association with Epstein-Barr virus latent membrane protein-1 (LMP-1). METHOD: The expressions of MMP-9, IL-8 and LMP-1 were immunohistochemical studied in 53 nasopharyngeal carcinoma sections. We statistically analyzed the correlation of these data and also the relationship between the clinical features and the experimental data of these patients. RESULT: The positive expression rate of MMP-9, IL-8 and LMP-1 were all 66.04% (35/53) and their average expression score were (33.19 +/- 29.73)%, (33.46 +/- 30.23)%, (35.49 +/- 29.63)% respectively. The expressions of MMP-9 and IL-8 both showed positive correlation with the expression of LMP-1 (r = 0.792, 0.786 respectively). The expressions of MMP-9, IL-8 and LMP-1 showed significant relationship with lymph nodes metastasis (P < 0.05), but no-significant association with age, gender, pathological classification, and clinical stage. CONCLUSION: The expressions of MMP-9, IL-8 and LMP-1 were significantly associated with neck lymph nodes metastasis in nasopharyngeal carcinoma. The correlation between LMP-1 and MMP-9, IL-8 showed LMP-1 might enhance neck lymph nodes metastasis by up-regulating the expressions of MMP-9 and IL-8.

We aimed at determining whether the expression of protease-activated receptor 2 (PAR-2) is involved in the progression of nasopharyngeal carcinoma (NPC) and correlated with latent membrane protein 1 (LMP-1), matrix metalloproteinases-9 (MMP9), and angiogenesis of tumor. PAR-2, LMP-1, and MMP9 expressions were detected in 57 biopsies of primary NPC by immunohistochemistry. The presence of Epstein-Barr virus (EBV) was determined using EBER in situ hybridization, and intratumoral microvessels were highlighted by staining endothelial cells for anti-CD34. The correlations with immunostainings and clinicopathological factors, as well as the follow-up data of patients, were analyzed statistically. Strong expression of PAR-2 in 61.4% (35/57) of the biopsies was correlated with extensive lymph node metastasis and advanced stage of NPC. The patients with PAR-2/LMP-1 or PAR-2/MMP9 dual high-expression tumors had a significant worse prognosis than those with single protein high expression and dual low or negative expression tumors (P=0.013 and 0.004, respectively). Angiogenesis in the tumor is related to overall survival of NPC patients (P=0.001), and exhibits strong PAR-2 expression or LMP-1 expression in tumors associated with increased intratumoral microvessel density (P=0.026 and 0.006, respectively). PAR-2 is a possible mediator cooperating with LMP-1 and MMP9 to influence the progression of NPC by inducing angiogenesis and promoting lymph node metastasis.

OBJECTIVE: To compare the expression of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 of nasopharyngeal carcinoma at Xi'an and Shenzhen area. METHOD: The protein expression of MMP 9 and TIMP 1 were studied by immunohistochemistry staining in 21 cases nasopharyngeal carcinoma of Xi'an and 22 cases of Shenzhen. RESULT: The expression positive rates of MMP-9 in nasopharyngeal carcinoma tissues at Xi'an and Shenzhen were 61.90% and 68.18%. The positive rates of TIMP-1 in nasopharyngeal carcinoma tissues at two regions were 28.57% and 31.82%. The expression between MMP-9 and TIMP-1 were negative relationship either Xi'an or Shenzhen. The expression of MMP-9 was positively correlated to lymph node metastasis, and TIMP-1 was no relationship with lymph node metastasis. The expression of MMP-9 and TIMP-1 was no difference between Xi'an and Shenzhen. CONCLUSION: The expression of MMP-9 and TIMP-1 in nasopharyngeal carcinoma 2 tissues was no difference between Xi'an and Shenzhen. Increase of MMP-9 play an important role in progression and metastasis of nasopharyngeal carcinoma.

OBJECTIVE: To test the expression of NF-kappaB/p65, MMP-3 and MMP-9 in nasopharyngeal carcinoma (NPC), and analyze their relationship and the clinical significance. METHOD: All samples were examined for the expression of NF-kappaB/p65, MMP-3 and MMP-9 by streptavidin-peroxidase immunohistochemistry. Chi2-test and Spearman-test were used to exam the correlation within them and the expression difference between nasopharyngeal carcinoma and normal nasopharyngeal mucosa. RESULT: Positive expression rate of NF-kappaB/p65 in nasopharyngeal carcinoma was 78.0%; positive expressions rate of MMP-3 and MMP-9 in nasopharyngeal carcinoma were 75.6%. There was positive relationship between NF-kappaB/p65, MMP-3, MMP-9 and N stage. The expression of NF-kappaB/p65 showed a significant positive correlation with the expression of MMP-3 and MMP-9. MMP-3 also had a significant positive correlation with MMP-9. CONCLUSION: The result suggests that NF-kappaB/p65, MMP-3 and MMP-9 are highly expressed in nasopharyngeal carcinoma cells. Their expression is correlated with lymph nodes metastasis, but do not have relationship with sex, age, pathological classification and clinical stage. Each of them has positive correlation with the others.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a type of neoplasm that is highly prevalent in East Asia and Africa with Epstein-Barr virus (EBV), genetic, and dietary factors implicated as possible aetiologic factors. Previous studies suggested the association of certain cytokines with the invasion and metastatic properties of NPC. The present study examined the roles of EBV latent membrane protein-1 (LMP1), interleukin-6 (IL-6), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-beta1) and laminin in the regulation of matrix-metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) in NPC. The effects of these factors on bmi-1, an oncogene, and ngx6, a tumour suppressor gene, were also investigated. METHODS: TW01 cells expressing LMP1 (TW01-LMP1) were established via transfection with the B95.8 EBV LMP1 gene. Both TW01 and TW01-LMP1 cells were treated with 100 pg/ml IL-6, 1000 pg/ml IL-10 and 100 pg/ml TGF-beta1, separately and also in combination at their respective concentration for 48 hours. Treated cells were subjected to laminin adherence assay. The cells were also cultured with and without laminin and assayed for MMP-3, MMP-9 and VEGF production using enzyme-linked immunosorbent assay (ELISA). The cellular apoptotic property was analysed using caspase-3 apoptosis assay. The expression of bmi-1 and ngx6 gene was investigated using real time reverse transcriptase polymerase chain reaction. RESULTS: LMP1 was found to reduce the adherence of NPC cells towards laminin (p < 0.05) as compared to control. Treatment with IL-6 at 100 pg/ml enhanced the production of MMP-9 in both TW01 and TW01-LMP1 cells (p < 0.05). When cultured on laminin, the levels of MMP-3 and VEGF were significantly increased (p < 0.05) in TW01-LMP1 cells. TW01-LMP1 cells had relatively greater resistance to apoptosis as compared to TW01 cells (p < 0.05). Laminin, IL-6 and LMP1 were found to up-regulate the expression of bmi-1 and suppressed the expression of ngx6. CONCLUSIONS: We conclude that IL-6 reduced cell adherence towards laminin and increased MMP-9 production in NPC cells. Our data suggested that EBV LMP1 was able to confer resistance of apoptosis and increased MMP-9 production in NPC cells. When cultured on laminin, TW01 cells expressing the EBV LMP1 (TW0-LMP1) that were treated with IL-6 at 100 pg/ml displayed increased MMP-9 production, up-regulation of bmi-1 oncogene expression and down-regulation of ngx6 tumour suppressor gene expression. These findings implicate the roles of EBV LMP1, laminin and IL-6 in the promotion of invasion and metastasis in NPC.

High-mobility group box 1 (HMGB1) is a versatile protein with intranuclear and extracellular functions. It is involved in invasion and metastasis in various human malignancies. However, the role of HMGB1 in non-small cell lung cancer (NSCLC) is unclear. We hypothesized that HMGB1 expression is a determinant of cellular invasiveness and metastasis in lung cancer. We examined HMGB1 expression in 48 NSCLC specimens by quantitative real-time PCR. High HMGB1 expression was significantly associated with clinically advanced stages (stage III-IV) (P < 0.05) and was correlated to expression of matrix metalloproteinase-9 (MMP-9) (P < 0.05). Patients with high levels of HMGB1 expression had poorer clinical prognosis. The expression level of MMP-9 and metastatic ability in vitro were significantly higher in an HMGB1-overexpressing human NSCLC cell lines (A549 and H23). The treatment with HMGB1 small interfering RNA reduced MMP-9 expression and the cellular metastatic ability in NSCLC cells. We also demonstrated that phosphoinositide 3-kinase/Akt and NF-kappaB-related pathways contributed to the HMGB1-induced MMP-9 expression and cellular metastatic ability.

KISS1 and matrix metalloproteinase-9 (MMP-9) may play important roles as metastasis suppressor and metastasis promoter genes, respectively, in a variety of malignancies. However, there is little information about their possible roles in non-small cell lung cancer (NSCLC). The goals of this study were to determine the mRNA and protein expressions of KISS1 and MMP-9 in NSCLC and their relations to metastasis and prognosis. The mRNA and protein expressions of KISS1 and of MMP-9 protein were detected by in situ hybridization and immunohistochemistry respectively in 85 cases of NSCLC, and their matched lymph node metastases. expressions of KISS1 mRNA and protein were significantly higher in low TNM stages of NSCLC (I-II) compared to more advanced stages (III-IV) (p<0.05). Moreover, in advanced TNM stages, cases without metastasis had higher KISS1 gene expression compared to those with lymph node metastasis (p<0.05). In contrast, MMP-9 expression was higher in stage III-IV NSCLC cases compared to stage I-II tumors (p<0.05) and higher in NSCLC cases with metastasis than those without metastasis (p<0.05). There was negative correction between KISS1 and MMP-9 protein expression (p<0.01). The 5-year survival rate in cases with higher KISS1 protein expression was significantly higher than in those with low expression (20.9 vs. 2.4%, p<0.01). However, the 5-year survival rate of patients with high MMP-9 protein expression were lower than those with low expression (19 vs. 4.7%, p<0.05). Our data suggest that KISS1 and MMP-9 may serve as potential prognostic and therapeutic markers in lung cancer.

BACKGROUND: It has been proven that CC chemokine receptor 7 (CCR7) is closely related with the lymph node metastasis of non-small cell lung cancer (NSCLC), but the mechanism of NSCLC metastasis is not very clear. The aim of this study is to investigate the expressions of CCR7 and MMP-9 in NSCLC and the relationship of their expressions, and to explore the mechanism of CCR7 promoting NSCLC metastasis. METHODS: The expressions of CCR7 and MMP-9 protein were detected in 90 specimens of human primary NSCLC by immunohistochemical SP method. Human large lung cell line BE1 cells were pre-incubated with CCL19 for 24 h; the changes of MMP-9 were detected by RT-PCR and Western blot. RESULTS: Immunohistochemistry showed that CCR7 was distributed in cytoplasm and/or membrane of tumor cells and MMP-9 was distributed in cytoplasm of cancer cells. The expressions of CCR7 and MMP-9 protein were found to be 70% (63/90) and 65.5% (59/90) in NSCLC, respectively. The expressions of CCR7 and MMP-9 protein were closely related to the clinical stages (P=0.003, P=0.001) and lymph node metastasis (P=0.004, P=0.003) of NSCLC, but there was no correlation with age, gender, histology (P > 0.05). Furthermore, a significant correlation was found between CCR7 and MMP-9 (r=0.342, P=0.001). In addition, the expressions of MMP-9 mRNA and protein levels were increased in CCL19 pre-incubated group (P < 0.05). CONCLUSIONS: The expressions of CCR7 and MMP-9 are significantly associated with NSCLC invasion and metastasis. The upregulation of MMP-9 is regulated by CCR7 in NSCLC.

PURPOSE: Cytochrome P450 (CYP) omega-hydroxylase, mainly consisting of CYP4A and CYP4F, converts arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) that induces angiogenic responses in vivo and in vitro. The present study examined the role of CYP omega-hydroxylase in angiogenesis and metastasis of human non-small cell lung cancer (NSCLC). METHODS: The effect of WIT003, a stable 20-HETE analog, on invasion was evaluated using a modified Boyden chamber in three NSCLC cell lines. A549 cells were transfected with CYP4A11 expression vector or exposed to CYP omega-hydroxylase inhibitor (HET0016) or 20-HETE antagonist (WIT002), and then omega-hydroxylation activity toward arachidonic acid and the levels of matrix metalloproteinases (MMPs) and VEGF were detected. The in vivo effects of CYP omega-hydroxylase were tested in established tumor xenografts and an experimental metastasis model in athymic mice. RESULTS: Addition of WIT003 or overexpression of CYP4A11 with an associated increase in 20-HETE production significantly induced invasion and expression of VEGF and MMP-9. Treatment of A549 cells with HET0016 or WIT002 inhibited invasion with reduction in VEGF and MMP-9. The PI3 K or ERK inhibitors also attenuated expression of VEGF and MMP-9. Compared with control, CYP4A11 transfection significantly increased tumor weight, microvessel density (MVD), and lung metastasis by 2.5-fold, 2-fold, and 3-fold, respectively. In contrast, WIT002 or HET0016 decreased tumor volume, MVD, and spontaneous pulmonary metastasis occurrences. CONCLUSION: CYP omega-hydroxylase promotes tumor angiogenesis and metastasis by upregulation of VEGF and MMP-9 via PI3 K and ERK1/2 signaling in human NSCLC cells.

Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrin-mediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP- and Src kinase-dependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad-rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination.

INTRODUCTION: Pancreatic cancer frequently is associated with venous invasion and hematogenous metastasis. AIMS: To determine morphologic features of invaded veins, intratumoral vascular composition, the correlation with liver metastasis, and expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9, and the mechanism of development of hematogenous metastasis. METHODOLOGY: We examined 32 patients with resected pancreatic cancer: 18 had postoperative liver metastasis, and 14 had no liver metastasis. Specimens were examined to determine the composition of veins and microvessels by staining of victoria-blue and CD34. We also investigated expression of VEGF, MMP-2, and MMP-9 by immunohistochemical staining. RESULTS: Venous invasion was detected in 31 of 32 patients. Invaded venous densities of middle- and large-sized veins were significantly higher in patients with liver metastasis than in those with nonliver metastasis, and they were related to MMP-2 and MMP-9 overexpression. Invaded veins with fragmentation of the lumen through cancer cells were considered to be an intravasation of cancer (destroyed type vein), and their numbers were significantly related to liver metastasis, and MMP-2 and MMP-9 overexpression. CONCLUSION: In conclusion, almost all the patients with pancreatic cancer showed venous invasion, indicating that invasion into large veins and destroyed type veins could be a risk factor for liver metastasis and that increased expression MMP-2 and MMP-9 were related to such invasion.

BACKGROUND/AIMS: Amphoterin is considered as a regulator for the ability of invasion and migration in tumor cells and embryonic neurons through binding to receptor for advanced glycation end products (RAGE), a multiligand cell surface molecule of the immunoglobulin superfamily. As matrix metalloproteinase-9 (MMP-9, gelatinase B) has been reported to play a critical role in tumor progression and metastasis, we have examined the relation of RAGE and MMP in human pancreatic cancer. METHODOLOGY: Three representative human pancreatic carcinoma cells were rendered for the study which show different metastatic potential, PANC-1 and MIA PaCa-2 as the cells with high ability, BxPC-3 as with low. The expression of RAGE was examined by RT-PCR. The expression of MMP-9 protein was examined by Western blotting. RESULTS: RAGE was strongly expressed in MIA PaCa-2 and PANC-1 that have high metastatic ability. On the contrary, RAGE was expressed little in BxPC-3 that has low ability. Similarly, expression of MMP-9 showed almost the same tendency. RAGE and MMP-9 are expressed concordant with the metastatic ability of the human pancreatic cancer cells. CONCLUSIONS: Control of these molecules could be a key to regulating the metastatic ability of pancreatic cancer and this may be exploited in targeted therapy of this cancer.

Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone) is an active constituent isolated from the root of Rheum palmatum L and is the main effective component of some Chinese herbs and plants. Pharmacological studies have demonstrated that emodin exhibits anti-cancer effects on several human cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. This study was performed to investigate the antiproliferative and antimetastatic effects of emodin on pancreatic cancer in vitro and in vivo. Our results showed that emodin induced a higher percentage of growth inhibition and apoptosis in the pancreatic cancer cell line SW1990 compared to that of control, and emodin suppressed the migration and invasion of SW1990 cells in a dose-dependent manner. To investigate the possible mechanisms involved in these events, we performed electrophoretic mobility shift assay (EMSA) and Western blot analysis, and found that emodin significantly down-regulated NF-kappaB DNA-binding activity, survivin and MMP-9 in SW1990 cells. Moreover, the expression of cleaved caspase-3 was up-regulated in SW1990 cells after treatment with emodin. In addition, a metastatic model simulating human pancreatic cancer was established by orthotopic implantation of histologically intact human tumor tissue into the pancreatic wall of nude mice. Oral administration of emodin significantly decreased tumor weight and metastasis compared to control. Furthermore, the expression of NF-kappaB, survivin and MMP-9 were also suppressed in tumor tissues after treatment with emodin. Collectively, our results indicated that emodin exerts antiproliferative and antimetastatic activity on pancreatic cancer both in vitro and in vivo, which may be related to down-regulation of NF-kappaB and its regulated molecules such as survivin and MMP-9 proteins. Consequently, these results provide important insights into emodin as an anti-invasive agent for the therapy of human pancreatic cancer.

BACKGROUND: Hematopoietic cells home to bone by means of chemo-attraction to marrow chemokines, and interaction of chemokines with their receptors leads to the expression/activation of adhesion molecules and proteases. Recent evidence suggests that similar mechanisms may be active in cancer metastasis. Previously, we showed that metalloproteases (MMPs), and in particular MMP-9, play a role in prostate cancer (PC) expansion in bone. METHODS: We used a variety of methods including RT-PCR, immunohistochemistry, ELISA, gelatin zymography, cellular motility and invasion, and subcellular fractionation of PC cells applied to in vivo and in vitro models. RESULTS: Here we showed that (a) CXCL12/CXCR4 axis is expressed in PC bone metastasis; (b) exogenous CXCL12 induced MMP-9 expression by PC cells; (c) bone stromal cells and bone tissue conditioned media induced the migration of PC cells in a CXCR4-dependent manner; (d) pharmacological inhibition of PI3 kinase and MAP kinase pathways abrogated CXCL12-induced MMP-9 expression and invasion of PC cells; (e) exogenous CXCL12 induced Akt1 phosphorylation is indispensable for proMMP-9 secretion, migration, and invasion of PC cells; (f) CXCR4 was localized to lipid rafts in PC cells and initiated Akt phosphorylation. CONCLUSIONS: These data suggest that chemoattractive mechanisms involve migration of cancer cells towards bone tissue, and that cell signaling induced by binding of the chemokine to its receptor leads to the activation of multiple signaling pathways and subsequent secretion of MMP-9 into the local environment. These findings provide a link between chemoattractive mechanisms, growth of tumor cells in bone, and tumor-enhanced bone matrix turnover.

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.

OBJECTIVE: The colorimetric in situ hybridization (CISH)-based matrix metalloproteinase (MMP)-to-E-cadherin (ECD) ratio (MER) has been revealed as an excellent marker for the disease stage in prostate cancer. The one aim of this study was investigating a new method for estimation of MER by bicolor fluorescent ISH (bicolor FISH) with a computerized fluorescence detector-based system. Another aim was examination of relation of MER by bicolor FISH with expression of vascular endothelial growth factor-C (VEGF-C). METHODS: The bicolor FISH technique used cyanin 5 (cy5)-labeled MMP-2 and -9 probes, and a cyanin 3 (cy3)-labeled ECD probe on needle biopsy specimens from 67 prostate cancer cases. The ISH was followed by computerized detection of the signal intensities and cy5-to-cy3 ratios using a fluorescence detector. VEGF-C expression was examined using cy5-labeled VEGF-C by computerized detection. RESULTS: The bicolor FISH-based MER was well correlated with CISH-based MER (P < 0.0001). The bicolor FISH-based MER correlated with Gleason score and pathologic stage of the cases. VEGF-C mRNA expression was associated with the pathologic stage and maximum lymph vessel density (LVD). The LVD was associated with VEGF-C expression at the tumor area where the maximum MER was detected (P < 0.0001). CONCLUSION: The MER was correlated with the VEGF-C expression and LVD, indicating lymph node metastasis of prostate cancer. Therefore, this computer-assisted MER is a useful marker for preoperative prediction of disease stage, especially lymph node metastasis, of prostate cancer.

OBJECTIVES: This study aims to analyze the clinicopathologic significance of stromal cell-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9) expression in human prostate cancer (CaP), and their involvement in the prognosis of CaP. MATERIALS AND METHODS: The expression of SDF-1, VEGF, and MMP-9 were measured using immunohistochemistry in 148 CaP patients who underwent radical prostatectomy for clinically localized disease and in 10 samples of benign prostatic hyperplasia (BPH). RESULTS: In the CaP group, VEGF and MMP-9 were more strongly expressed in the tumor cells compared with the BPH group. High intensity SDF-1, VEGF, and MMP-9 stains in tumor areas strongly correlated with lymph node metastasis, pathologic stage, and differentiation. Univariate and multivariate analysis showed that SDF-1, VEGF, and lymph node metastasis were independent prognostic factors for prostate cancer-specific survival. High levels of MMP-9, pathologic stage, and differentiation were associated with prostate cancer-specific survival in univariate analysis but the risk estimate was not significant in multivariate analysis. CONCLUSIONS: High expression levels of SDF-1, VEGF, and MMP-9 are more correlated with lymph node metastatic prostate carcinoma compared with non-lymph-node metastatic cancer. High expression levels of SDF-1 and VEGF strongly predict the biochemical progression in CaP patients after radical prostatectomy.

Although the potential roles of endothelial cells in the microvascules of prostate cancer during angiogenesis have been documented, their direct impacts on the prostate cancer metastasis remain unclear. We found that the CD31-positive and CD34-positive endothelial cells are increased in prostate cancer compared with the normal tissues and that these endothelial cells were decreased upon castration, gradually recovered with time, and increased after prostate cancer progressed into the castration-resistant stage, suggesting a potential linkage of these endothelial cells with androgen deprivation therapy. The in vitro invasion assays showed that the coculture of endothelial cells with prostate cancer cells significantly enhanced the invasion ability of the prostate cancer cells. Mechanism dissection found that coculture of prostate cancer cells with endothelial cells led to increased interleukin (IL)-6 secretion from endothelial cells, which may result in downregulation of androgen receptor (AR) signaling in prostate cancer cells and then the activation of TGF-beta/matrix metalloproteinase-9 (MMP-9) signaling. The consequences of the IL-6-->AR-->TGFbeta-->MMP-9 signaling pathway might then trigger the increased invasion of prostate cancer cells. Blocking the IL-6-->AR-->TGFbeta-->MMP-9 signaling pathway either by IL-6 antibody, AR-siRNA, or TGF-beta1 inhibitor all interrupted the ability of endothelial cells to influence prostate cancer invasion. These results, for the first time, revealed the important roles of endothelial cells within the prostate cancer microenvironment to promote the prostate cancer metastasis and provide new potential targets of IL-6-->AR-->TGFbeta-->MMP-9 signals to battle the prostate cancer metastasis.

Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysis-defined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-kappaB (NF-kappaB) site. Mutating this NF-kappaB binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-kappaB binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation.

Head and neck squamous cell carcinomas (HNSCC) are characterized by a marked propensity for local invasion and cervical lymph node metastasis. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR), c-erbB-2, vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in tumor samples of 91 HNSCC patients, and to study a possible correlation to various clinico-pathologic parameters. The expression of EGFR, c-erbB-2, VEGF, MMP-2, -3 and -9 was analyzed in the same paraffin embedded tissue by semi-quantitative immunohistochemical staining. High expression of EGFR, c-erbB-2, MMP-2 or -9 was associated with advanced clinical stages, nodal metastases and tumor-stages. However, high expression of VEGF or MMP-3 was not associated with any clinico-pathologic parameters except significant correlation between VEGF and the tumor site. There were significant correlations between EGFR, c-erbB-2, MMP-2 and -9 in HNSCC patients. Conversely, no correlation was found between VEGF or MMP-3 and the other markers. However, significant correlation was found between MMP-3 or -9 and VEGF. The results indicate that the expression of EGFR, c-erbB-2, VEGF or MMPs play an important role in tumor growth, invasion and metastasis in HNSCC. The authors conclude that EGFR, c-erbB-2, MMP-2 and -9 could be good independent prognostic markers, but not VEGF and MMP-3.

Matrix metalloproteinase 9 (MMP9) and CD147 play a role in invasion and metastasis of many types of human malignancies. The correlation of the expression of MMP9 and CD147 with invasion and metastasis of invasive squamous cell carcinoma (SCC) of the uterine cervix has not been examined. In the present study, RT-PCR assay was used to detect the expression level of MMP9 mRNA semiquantitatively, and immunohistochemical stain was adapted to evaluate the score of CD147 on the cell membrane or in the cytoplasm of tumor cells of 65 cases of invasive squamous cell carcinoma of the uterine cervix and 21 cases of chronic cervitis tissues. MMP9 and CD147 expression in correlation with invasion, metastasis, and differentiation of invasive SCC of the uterine cervix was analyzed statistically. We found that MMP9 and CD147 expression was elevated significantly in tumor tissue compared to the control (cervical epithelium of chronic cervitis) (P<0.01). In the comparison of MMP9 and CD147 expression in 47 cases with lymph node metastasis and 18 cases without lymph node metastasis, there was a significantly higher expression of MMP9 and CD147 in the group with lymph node metastasis (P<0.05 for MMP9, P<0.01 for CD147). MMP9 expression was significantly higher in 24 cases of poor differentiation than in 41 cases of moderate differentiation (P<0.05). No difference was found in CD147 expression between poor and moderate differentiation (P>0.05). No significant difference in MMP9 and CD147 expression levels was obtained between 26 cases of FIGO stage I tumors and 39 cases of stage II tumors (P>0.05 for MMP9, P>0.05 CD147). There was no correlation between MMP9 or CD147 expression levels and the resected tumor size (P>0.05). The positive correlation (r=0.568, P<0.001) of MMP9 expression and CD147 score was seen in the tumor tissues of 65 cases. The data in this study show that MMP9 and CD147 expression are correlated with invasion, metastasis of squamous cell carcinoma of the uterine cervix, and that MMP9 expression is correlated with poor differentiation of invasive squamous cell carcinoma of the uterine cervix.

OBJECTIVE: Reversion-inducing cysteine-rich protein with kazal motifs (RECK) may negatively regulate matrix metalloproteinase 9 (MMP9) activity and suppress tumor invasion and metastasis. The aim of this study was to investigate whether RECK and MMP were involved in regulating middle ear carcinoma invasion and metastasis. METHODS: RECK and MMP9 were measured in 30 middle ear squamous cell carcinoma tissues and 20 adjacent normal external ear canal skin tissues using immunohistochemical analysis. RESULTS: The positive rate of RECK expression in the middle ear squamous cell carcinoma was much lower than that in the normal external ear canal skin. In contrast, the positive rate of MMP9 expression was higher in middle ear squamous cell carcinoma tissue than that in normal external ear canal skin tissue. The expressions of RECK and MMP9 were correlated with the histological grade and tumor stage, but not with patient age or gender. CONCLUSIONS: RECK and MMP9 are involved in middle ear squamous cell carcinoma and may serve as markers to evaluate progression and metastasis.

Matrix metalloproteinases (MMPs) are elevated in patients with squamous cell carcinoma (SCC) over either the head and neck (HNSCC) or the esophagus (ESCC). Synchronous SCC with both HNSCC and ESCC predispose to worse survival. This study tested if serum MMP levels correlate with clinical features and predict survival for HNSCC, ESCC, and synchronous SCC. One hundred and thirty patients with SCCs in upper aero-digestive tract (70 ESCC, 20 HNSCC, and 40 synchronous SCC) and 74 healthy controls were assessed for serum MMP-3, -7, and -9 titers by enzyme-linked immunosorbent assay. The titers were validated to their correlations to clinical features and survival rates of the different SCC groups. Patients with SCCs had significantly higher serum MMP-3, -7, and -9 titers than the controls (P < 0.001) but there was no difference among the three SCC groups. Based on the optimal MMP cut-off values by ROC curve, elevated MMP-3 and MMP-9, but not MMP-7, correlated with distant metastasis and poor survival (P < 0.05). Concomitantly elevated MMP-3 (>14 ng/mL) and MMP-9 (>329.3 ng/mL) independently correlated with poor two-year survival (P = 0.002, by log rank test). Cox regression confirmed that such concomitant elevation was superior to the tumor stage of either ESCC or HNSCC in predicting survival for synchronous SCC. Serum MMPs are elevated in SCC of the upper aero-digestive tract. Especially for synchronous SCC, concomitantly elevated MMP-3 and MMP-9 levels serve as better biomarkers to predict prognosis than TNM staging of ESCC or HNSCC.

The mechanisms leading to squamous cell carcinoma of head and neck (SCCHN) metastasis are not fully understood. Although evidence shows that the chemokine receptor 7 (CCR7) and its ligand CCL19 may regulate tumor dissemination, their role is not clearly defined in SCCHN. Matrix metalloproteinases break consisting of tissue barrier to the surrounding tissue invasion and metastasis by destroying the balance of matrix degradation of the basement membrane of tumor cells and extracellular matrix (ECM). We used chemotaxis and migration assays, western blotting, gelatin zymography, actin polymerization assay, immunofluorescence staining and immunohistochemical analysis to explore whether MMP-9 can be activated by CCL19 (CCR7's ligand) and its role in SCCHN. The experiments were performed in the metastatic SCCHN cell line PCI-37B after pre-incubation of the cells with CCL19 and SB-3CT (inhibitor of MMP-9). Our results demonstrated that CCR7 favors PCI-37B cell chemotaxis and migration, upregulation of MMP-9 protein and motivates the activity of MMP-9 protein, induces reorganization of the actin cytoskeleton and upregulation of MMP-9 protein. SB-3CT can block all these effects. Collectively, our data indicated that CCR7 regulates cell chemotaxis and migration via MMP-9 in metastatic SCCHN, and these results provide a basis for new strategies in preventing metastases of SCCHN.

BACKGROUND: Squamous cell carcinoma of the tongue has frequent lymph node metastases and poor prognosis. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are reported to degrade basement membrane,facilitating invasion and metastasis. This study determined the expression of MMP-2/MMP-9 in primary tongue cancer with or without cervical metastases and analysed the significance of such expression in relation to the presence or absence of metastases. METHODS: expressions of MMP-2MMP-9 were detected by immunohistochemistry in 10 specimens of normal oral mucosa, 20 lymph node-negative tongue cancers, 41 lymph node-positive tongue cancers and their metastasized tumours in cervical lymph nodes. RESULTS: MMP-2MMP-9 expression was seldom found in normal epithelium. In lymph node-negative tongue cancer, 45% and 40% of these primary tumours were positively stained for MMP-2MMP-9, respectively. Importantly, in lymph node-positive tongue cancer, 71% and 79% of these primary tumours were positive for MMP-2MMP-9, respectively. Overexpression of MMP-2MMP-9 was present in the metastatic lymph nodes. CONCLUSIONS: Our results imply that MMP-2 and/or MMP-9 play an important role in invasion and metastasis in tongue cancer, and that analysis of MMP expression and/or activity in primary tumours may have a predictive value for the actual or potential presence of cervical metastases.

Some studies show the Notch signaling pathway may participate in carcinoma invasion and metastasis. However, the mechanisms by which Notch1 mediates cell invasion and migration, especially in lingual squamous cell carcinoma, are not yet known. In the current study, we demonstrated for the first time the anti-invasion and anti-metastasis effect of down-regulation of Notch1 in lingual squamous cell carcinoma. Down-regulation of Notch1 could be an effective approach for inhibition of the expression of matrix metalloproteinase (MMP)-2 and MMP-9 resulting in the inhibition of invasion and metastasis, which could be useful for devising novel preventive and therapeutic strategies for lingual squamous cell carcinoma.